Human monoclonal antibodies to programmed death ligand 1 (PD-L1)

ABSTRACT

The present disclosure provides isolated monoclonal antibodies, particularly human monoclonal antibodies that specifically bind to PD-L1 with high affinity. Nucleic acid molecules encoding the antibodies of this disclosure, expression vectors, host cells and methods for expressing the antibodies of this disclosure are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The disclosure also provides methods for detecting PD-L1, as well as methods for treating various diseases, including cancer and infectious diseases, using anti-PD-L1 antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/807,522, filed Jul. 23, 2015, which is a divisional of U.S.application Ser. No. 13/746,773, filed Jan. 22, 2013, now U.S. Pat. No.9,102,725, which is a divisional application of U.S. application Ser.No. 13/091,936, filed Apr. 21, 2011, now issued as U.S. Pat. No.8,383,796, which is a divisional application of U.S. application Ser.No. 11/917,727, having 371(c) date of Jun. 9, 2008, now issued as U.S.Pat. No. 7,943,743, which is a national phase of PCT Appl. No.PCT/US2006/026046, filed Jun. 30, 2006, which claims the benefit of U.S.Provisional Patent Application No. 60/696,426, filed Jul. 1, 2005, eachof which is incorporated herein by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted in ASCII text file format via EFS-Web and is herebyincorporated by reference in its entirety. Said ASCII copy, created onJun. 17, 2016, is named “3338_0370006_SeqListing_ST25.txt”, and is77,664 bytes in size.

BACKGROUND

Programmed death 1 (PD-1) is a member of the CD28 family of receptors,which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The initial membersof the family, CD28 and ICOS, were discovered by functional effect onaugmenting T cell proliferation following the addition of monoclonalantibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al.(1980) Immunogenics 10:247-260). Two cell surface glycoprotein ligandsfor PD-1 have been identified, PD-L1 and PD-L2, and have been shown todownregulate T cell activation and cytokine secretion upon binding toPD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al.(2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 2:634-43;Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). Both PD-L1 (B7-H1)and PD-L2 (B7-DC) are B7 homologs that bind to PD-1, but do not bind toother CD28 family members (Blank et al. (2004). Expression of PD-L1 onthe cell surface has also been shown to be upregulated through IFN-γstimulation.

PD-L1 expression has been found in several murine and human cancers,including human lung, ovarian and colon carcinoma and various myelomas(Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin CancerRes 11:2947-53). PD-L1 has been suggested to play a role in tumorimmunity by increasing apoptosis of antigen-specific T-cell clones (Donget al. (2002) Nat Med 8:793-800). It has also been suggested that PD-L1might be involved in intestinal mucosal inflammation and inhibition ofPD-L1 suppresses wasting disease associated with colitis (Kanai et al.(2003) J Immunol 121:4156-63).

SUMMARY

The present invention provides isolated monoclonal antibodies, inparticular human monoclonal antibodies that bind to PD-L1 and exhibitnumerous desirable properties. These properties include high affinitybinding to human PD-L1. Still further, antibodies of the invention havebeen shown to increase T-cell proliferation, IFN-γ secretion, and IL-2secretion in a mixed lymphocyte reaction.

In one aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof wherein the antibodyexhibits at least one of the following properties:

-   -   (a) binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M or less;    -   (b) increases T-cell proliferation in a mixed lymphocyte        reaction (MLR) assay;    -   (c) increases interferon-γ production in an MLR assay;    -   (d) increases IL-2 secretion in an MLR assay;    -   (e) stimulates antibody responses; or    -   (f) reverses the effect of T regulatory cells on T cell effector        cells and/or dendritic cells.        Preferably the antibody is a human antibody, although in        alternative embodiments the antibody can be, for example, a        murine antibody, a chimeric antibody or humanized antibody.

In particular embodiments, the antibody binds to human PD-L1 with aK_(D) of 5×10⁻⁸ M or less, binds to human PD-L1 with a K_(D) of 1×10⁻⁸ Mor less, binds to human PD-L1 with a K_(D) of 5×10⁻⁸ M or less, binds tohuman PD-L1 with a K_(D) of 5×10⁻⁹ M or less, or binds to human PD-L1with a K_(D) of between 1×10-M and 1×10⁻¹⁰ M.

In another embodiment, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, wherein the antibodycross-competes for binding to PD-L1 with a reference antibodycomprising:

-   -   (a) the human heavy chain variable region comprises an amino        acid sequence selected from the group consisting of SEQ ID        NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and    -   (b) the human light chain variable region comprises an amino        acid sequence selected from the group consisting of SEQ ID        NOs:11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.        In various embodiments, the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:11;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:2; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:12;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:13;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:4; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:14;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:5; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:15;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:6; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:16;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:7; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:17;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:8; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 18;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:9; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO-19;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:10; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:20.

In another aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof comprising a heavy chainvariable region that is the product of or derived from a human V_(H)1-18 gene, wherein the antibody specifically binds PD-L1. The inventionfurther provides an isolated monoclonal antibody, or an antigen-bindingportion thereof; comprising a heavy chain variable region that is theproduct of or derived from a human V_(H) 1-69 gene, wherein the antibodyspecifically binds PD-L1. The invention farther provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising aheavy chain variable region that is the product of or derived from ahuman V_(H) 1-3 gene, wherein the antibody specifically binds PD-L1. Theinvention further provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a heavy chain variableregion that is the product of or derived from a human V_(H) 3-9 gene,wherein the antibody specifically binds PD-L1. The invention furtherprovides an isolated monoclonal antibody, or an antigen-binding portionthereof, comprising a light chain variable region that is the product ofor derived from a human V_(K) L6 gene, wherein the antibody specificallybinds PD-L1. The invention further provides an isolated monoclonalantibody, or an antigen-binding portion thereof; comprising a lightchain variable region that is the product of or derived from a humanV_(K) L15 gene, wherein the antibody specifically binds PD-L1. Theinvention further provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) A27 gene,wherein the antibody specifically binds PD-L1. The invention furtherprovides an isolated monoclonal antibody, or an antigen-binding portionthereof; comprising a light chain variable region that is the product ofor derived from a human V, L18 gene, wherein the antibody specificallybinds PD-L1.

In a particularly preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereof;comprising:

-   -   (a) a heavy chain variable region of a human V_(H) 1-18 gene;        and    -   (b) a light chain variable region of a human V_(K) L6 gene;    -   wherein the antibody specifically binds to PD-L1.

In another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof comprising:

-   -   (a) a heavy chain variable region of a human V_(H) 1-69 gene;        and    -   (b) a light chain variable region of a human V_(K) L6 gene;    -   wherein the antibody specifically binds to PD-L1.

In another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof comprising:

-   -   (a) a heavy chain variable region of a human V_(H) 1-3 gene; and    -   (b) a light chain variable region of a human V_(K) L15 gene;    -   wherein the antibody specifically binds to PD-L1.

In another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof comprising:

-   -   (a) a heavy chain variable region of a human V_(H) 1-69 gene;        and    -   (b) a light chain variable region of a human V_(K) L15 gene;    -   wherein the antibody specifically binds to PD-L1.

In another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof; comprising:

-   -   (a) a heavy chain variable region of a human V_(H) 3-9 gene; and    -   (b) a light chain variable region of a human V_(K) L15 gene;    -   wherein the antibody specifically binds to PD-L1.

In another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof; comprising:

-   -   (a) a heavy chain variable region of a human VH 3-9 gene; and    -   (b) a light chain variable region of a human V_(K) L18 gene;    -   wherein the antibody specifically binds to PD-L1.

In another aspect, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, comprising:

-   -   a heavy chain variable region that comprises CDR1, CDR2, and        CDR3 sequences; and a light chain variable region that comprises        CDR1, CDR2, and CDR3 sequences, wherein:    -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of SEQ ID        NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, and conservative        modifications thereof;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of SEQ ID        NOs:71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, and conservative        modifications thereof; and    -   (c) the antibody specifically binds to human PD-L1.        Preferably, the heavy chain variable region CDR2 sequence        comprises an amino acid sequence selected from the group        consisting of amino acid sequences of SEQ ID NOs:31, 32, 33, 34,        35, 36, 37, 38, 39, and 40, and conservative modifications        thereof; and the light chain variable region CDR2 sequence        comprises an amino acid sequence selected from the group        consisting of amino acid sequences of SEQ ID NOs:61, 62, 63, 64,        65, 66, 67, 68, 69, and 70, and conservative modifications        thereof. Preferably, the heavy chain variable region CDR1        sequence comprises an amino acid sequence selected from the        group consisting of amino acid sequences of SEQ ID NOs:21, 22,        23, 24, 25, 26, 27, 28, 29, and 30, and conservative        modifications thereof; and the light chain variable region CDR1        sequence comprises an amino acid sequence selected from the        group consisting of amino acid sequences of SEQ ID NOs:51, 52,        53, 54, 55, 56, 57, 58, 59, and 60, and conservative        modifications thereof.

In yet another aspect, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof; comprising a heavy chainvariable region and a light chain variable region, wherein:

-   -   (a) the heavy chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs:1, 2,        3, 4, 5, 6, 7, 8, 9, and 10;    -   (b) the light chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs:11,        12, 13, 14, 15, 16, 17, 18, 19, and 20; and    -   (c) the antibody binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M        or less.

In a preferred embodiment, the antibodies additionally comprise at leastone of the following properties:

-   -   (a) the antibody increases T-cell proliferation in a mixed        lymphocyte reaction (MLR) assay;    -   (b) the antibody increases interferon-γ production in an MLR        assay; or    -   (c) the antibody increases IL-2 secretion in an MLR assay.

In preferred embodiments, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof comprising:

-   -   (a) a heavy chain variable region CDR1 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:21,        22, 23, 24, 25, 26, 27, 28, 29, and 30;    -   (b) a heavy chain variable region CDR2 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:31,        32, 33, 34, 35, 36, 37, 38, 39, and 40;    -   (c) a heavy chain variable region CDR3 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:41,        42, 43, 44, 45, 46, 47, 48, 49, and 50;    -   (d) a light chain variable region CDR1 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:51,        52, 53, 54, 55, 56, 57, 58, 59, and 60;    -   (e) a light chain variable region CDR2 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:61,        62, 63, 64, 65, 66, 67, 68, 69, and 70; and    -   (f) a light chain variable region CDR3 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:71,        72, 73, 74, 75, 76, 77, 78, 79, and 80;    -   wherein the antibody specifically binds PD-L1.        A preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:21;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:31;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:41;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:51;    -   (c) a light chain variable region CDR2 comprising SEQ ID NO:61;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:71.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:22;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:32;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:42;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:52;    -   (c) a light chain variable region CDR2 comprising SEQ ID NO:62;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:72.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:23;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:33;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:43;    -   (d) a light chain variable region CDR1 comprising SEQ II) NO:53;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:63;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:73.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:24;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:34;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:44;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:54;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:64;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:74.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:25;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:35;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:45;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:55;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:65;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID) NO:75.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:26;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:36;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:46;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:56;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:66;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:76.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:27;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:37;    -   (o) a heavy chain variable region CDR3 comprising SEQ ID NO:47;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:57;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:67;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:77.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:28;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:38;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:48;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:58;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:68;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:78.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:29;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:39;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:49;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:59;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:69;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:79.        Another preferred combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO:30;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO:40;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO:50;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO:60;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO:70;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO:80.        Other preferred antibodies of the invention, or antigen binding        portions thereof comprise:    -   (a) a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:1, 2,        3, 4, 5, 6, 7, 8, 9, and 10; and    -   (b) a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:11,        12, 13, 14, 15, 16, 17, 18, 19, and 20; wherein the antibody        specifically binds PD-L1.        A preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:11.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:2; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:12.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID) NO:3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:13.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:4; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:14.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:5; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:15.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:6; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:16.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:7; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:17.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO-8; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:18.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:9; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:19.        Another preferred combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:10; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO:20.

In another aspect of the instant disclosure, antibodies, orantigen-binding portions thereof, are provided that compete for bindingto PD-L1 with any of the aforementioned antibodies.

The antibodies of the instant disclosure can be, for example,full-length antibodies, for example of an IgG1 or IgG4 isotype.Alternatively, the antibodies can be antibody fragments, such as Fab orFab′2 fragments, or single chain antibodies.

The instant disclosure also provides an immunoconjugate comprising anantibody of the invention, or antigen-binding portion thereof, linked toa therapeutic agent, such as a cytotoxin or a radioactive isotope. Theinvention also provides a bispecific molecule comprising an antibody, orantigen-binding portion thereof, of the invention, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen binding portion thereof.

Compositions comprising an antibody, or antigen-binding portion thereof;or immunoconjugate or bispecific molecule of the instant disclosure anda pharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of the invention are also encompassed by theinvention, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Moreover, theinvention provides a transgenic mouse comprising human immunoglobulinheavy and light chain transgenes, wherein the mouse expresses anantibody of the invention, as well as hybridomas prepared from such amouse, wherein the hybridoma produces the antibody of the invention.

In yet another aspect, the invention provides a method of modulating animmune response in a subject comprising administering to the subject theantibody, or antigen-binding portion thereof; of the invention such thatthe immune response in the subject is modulated. Preferably, theantibody of the invention enhances, stimulates or increases the immuneresponse in the subject.

In a further aspect, the invention provides a method of inhibitinggrowth of tumor cells in a subject, comprising administering to asubject a therapeutically effective amount of an anti-PD-L1 antibody, orantigen-binding portion thereof. The antibodies of the invention arepreferred for use in the method although other anti-PD-L1 antibodies canbe used instead (or in combination with an anti-PD-L1 antibody of theinvention). For example, a chimeric, humanized or fully human anti-PD-L1antibody can be used in the method of inhibiting tumor growth.

In a further aspect, the invention provides a method of treating aninfectious disease in a subject, comprising administering to a subject atherapeutically effective amount of an anti-PD-L1 antibody, orantigen-binding portion thereof. The antibodies of the invention arepreferred for use in the method although other anti-PD-L1 antibodies canbe used instead (or in combination with an anti-PD-L1 antibody of theinvention). For example, a chimeric, humanized or fully human anti-PD-L1antibody can be used in the method of treating an infectious disease.

Still further, the invention provides a method of enhancing an immuneresponse to an antigen in a subject, comprising administering to thesubject: (I) the antigen; and (ii) an anti-PD-L1 antibody, orantigen-binding portion thereof, such that an immune response to theantigen in the subject is enhanced. The antigen can be, for example, atumor antigen, a viral antigen, a bacterial antigen or an antigen from apathogen. The antibodies of the invention are preferred for use in themethod although other anti-PD-L1 antibodies can be used instead (or incombination with an anti-PD-L1 antibody of the invention). For example,a chimeric, humanized or fully human anti-PD-L1 antibody can be used inthe method of enhancing an immune response to an antigen in a subject.

The invention also provides methods for making “second generation”anti-PD-L1 antibodies based on the sequences of the anti-PD-L1antibodies provided herein. For example, the invention provides a methodfor preparing an anti-PD-L1 antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence that is selected from the group consisting ofSEQ ID NOs:21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, a CDR2 sequencethat is selected from the group consisting of SEQ ID NOs:31, 32, 33, 34,35, 36, 37, 38, 39, and 40; and a CDR3 sequence that is selected fromthe group consisting of SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49,and 50; or (ii) a light chain variable region antibody sequencecomprising a CDR1 sequence that is selected from the group consisting ofSEQ ID NOs:51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, a CDR2 sequencethat is selected from the group consisting of SEQ ID NOs:61, 62, 63, 64,65, 66, 67, 68, 69, and 70, and a CDR3 sequence that is selected fromthe group consisting of SEQ ID NOs:71, 72, 73, 74, 75, 76, 77, 78, 79,and 80;

(b) altering at least one amino acid residue within at least onevariable region antibody sequence, said sequence being selected from theheavy chain variable region antibody sequence and the light chainvariable region antibody sequence, to create at least one alteredantibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all references, Genbank entries,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A shows the nucleotide sequence (SEQ ID NO:81) and amino acidsequence (SEQ ID NO:1) of the heavy chain variable region of the 3G10human monoclonal antibody. The CDR1 (SEQ ID NO:21), CDR2 (SEQ ID NO:31)and CDR3 (SEQ ID NO:41) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO:91) and amino acidsequence (SEQ ID NO: 11) of the light chain variable region of the 3G10human monoclonal antibody. The CDR1 (SEQ ID NO:51), CDR2 (SEQ ID NO:61)and CDR3 (SEQ ID NO:71) regions are delineated and the V and J germlinederivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO:82) and amino acidsequence (SEQ ID NO:2) of the heavy chain variable region of the 12A4human monoclonal antibody. The CDR1 (SEQ ID NO:22), CDR2 (SEQ ID NO:32)and CDR3 (SEQ ID NO:42) regions are delineated and the V and J germlinederivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO:92) and amino acidsequence (SEQ ID NO: 12) of the light chain variable region of the 12A4human monoclonal antibody. The CDR1 (SEQ ID NO:52), CDR2 (SEQ ID NO:62)and CDR3 (SEQ ID NO:72) regions are delineated and the V and J germlinederivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO:83) and amino acidsequence (SEQ ID NO:3) of the heavy chain variable region of the 10A5human monoclonal antibody. The CDR1 (SEQ ID NO:23), CDR2 (SEQ ID NO:33)and CDR3 (SEQ ID NO:43) regions are delineated and the V and J germlinederivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO:93) and amino acidsequence (SEQ ID NO: 13) of the light chain variable region of the 10A5human monoclonal antibody. The CDR1 (SEQ ID NO:53), CDR2 (SEQ ID NO:63)and CDR3 (SEQ ID NO:73) regions are delineated and the V and J germlinederivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO:84) and amino acidsequence (SEQ ID NO:4) of the heavy chain variable region of the 5F8human monoclonal antibody. The CDR1 (SEQ ID NO:24), CDR2 (SEQ ID NO:34)and CDR3 (SEQ ID NO:44) regions are delineated and the V and J germlinederivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO-94) and amino acidsequence (SEQ ID NO: 14) of the light chain variable region of the 5F8human monoclonal antibody. The CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:64)and CDR3 (SEQ ID NO:74) regions are delineated and the V and J germlinederivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO:85) and amino acidsequence (SEQ ID NO:5) of the heavy chain variable region of the 10H10human monoclonal antibody. The CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:35)and CDR3 (SEQ ID NO:45) regions are delineated and the V and J germlinederivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO:95) and amino acidsequence (SEQ ID NO:15) of the light chain variable region of the 10H10human monoclonal antibody. The CDR1 (SEQ ID NO:55), CDR2 (SEQ ID NO:65)and CDR3 (SEQ ID NO:75) regions are delineated and the V and J germlinederivations are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO:86) and amino acidsequence (SEQ ID NO:6) of the heavy chain variable region of the 1B12human monoclonal antibody. The CDR1 (SEQ ID NO:26), CDR2 (SEQ ID NO:36)and CDR3 (SEQ ID NO:46) regions are delineated and the V and J germlinederivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO:96) and amino acidsequence (SEQ ID NO: 16) of the light chain variable region of the 1B12human monoclonal antibody. The CDR1 (SEQ ID NO:56), CDR2 (SEQ ID NO:66)and CDR3 (SEQ ID NO:76) regions are delineated and the V and J germlinederivations are indicated.

FIG. 7A shows the nucleotide sequence (SEQ ID NO:87) and amino acidsequence (SEQ ID NO:7) of the heavy chain variable region of the 7H1human monoclonal antibody. The CDR1 (SEQ ID NO:27), CDR2 (SEQ ID NO:37)and CDR3 (SEQ ID NO:47) regions are delineated and the V and J germlinederivations are indicated.

FIG. 7B shows the nucleotide sequence (SEQ ID NO:97) and amino acidsequence (SEQ ID NO:17) of the light chain variable region of the 7H1human monoclonal antibody. The CDR1 (SEQ ID NO:57), CDR2 (SEQ ID NO:67)and CDR3 (SEQ ID NO:77) regions are delineated and the V and J germlinederivations are indicated.

FIG. 8A shows the nucleotide sequence (SEQ ID NO:88) and amino acidsequence (SEQ ID NO:8) of the heavy chain variable region of the 11E6human monoclonal antibody. The CDR1 (SEQ ID) NO:28), CDR2 (SEQ ID NO:38)and CDR3 (SEQ ID NO:48) regions are delineated and the V and J germlinederivations are indicated.

FIG. 8B shows the nucleotide sequence (SEQ ID NO:98) and amino acidsequence (SEQ ID NO 18) of the light chain variable region of the 11E6human monoclonal antibody. The CDR1 (SEQ ID NO:58), CDR2 (SEQ ID NO:68)and CDR3 (SEQ ID NO:78) regions are delineated and the V and J germlinederivations are indicated.

FIG. 9A shows the nucleotide sequence (SEQ ID NO:89) and amino acidsequence (SEQ ID NO:9) of the heavy chain variable region of the 12B7human monoclonal antibody. The CDR1 (SEQ ID NO:29), CDR2 (SEQ ID NO:39)and CDR3 (SEQ ID NO:49) regions are delineated and the V and J germlinederivations are indicated.

FIG. 9B shows the nucleotide sequence (SEQ ID NO:99) and amino acidsequence (SEQ ID NO: 19) of the light chain variable region of the 12B7human monoclonal antibody. The CDR1 (SEQ ID NO:59), CDR2 (SEQ ID NO:69)and CDR3 (SEQ ID NO:79) regions are delineated and the V and J germlinederivations are Indicated.

FIG. 10A shows the nucleotide sequence (SEQ ID NO:90) and amino acidsequence (SEQ ID NO: 10) of the heavy chain variable region of the 13G4human monoclonal antibody. The CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:40)and CDR3 (SEQ ID NO:50) regions are delineated and the V and J germlinederivations are indicated.

FIG. 10B shows the nucleotide sequence (SEQ ID NO:100) and amino acidsequence (SEQ ID NO:20) of the light chain variable region of the 13G4human monoclonal antibody. The CDR1 (SEQ ID NO:60), CDR2 (SEQ ID NO:70)and CDR3 (SEQ ID NO:80) regions are delineated and the V and J germlinederivations are indicated.

FIG. 11 shows the alignment of the amino acid sequence of the heavychain variable region of 3G10 with the human germline V_(H) 1-18 aminoacid sequence (SEQ ID NO:101).

FIG. 12 shows the alignment of the amino acid sequence of the heavychain variable region of 12A4 with the human germline V_(H) 1-69 aminoacid sequence (SEQ ID NO:102).

FIG. 13 shows the alignment of the amino acid sequence of the heavychain variable region of 10A5 with the human germline V_(H) 1-3 aminoacid sequence (SEQ ID NO:103).

FIG. 14 shows the alignment of the amino acid sequence of the heavychain variable region of 5F8 with the human germline V_(H) 1-69 aminoacid sequence (SEQ ID NO:102).

FIG. 15 shows the alignment of the amino acid sequence of the heavychain variable region of 10H10 with the human germ line V_(H) 3-9 aminoacid sequence (SEQ ID NO:104).

FIG. 16 shows the alignment of the amino acid sequence of the heavychain variable region of 1B12 with the human germline V_(H) 1-69 aminoacid sequence (SEQ ID NO:102).

FIG. 17 shows the alignment of the amino acid sequence of the heavychain variable region of 7H1 with the human germline V_(H) 1-69 aminoacid sequence (SEQ ID NO:102).

FIG. 18 shows the alignment of the amino acid sequence of the heavychain variable region of 11E6 with the human germline V_(H) 1-69 aminoacid sequence (SEQ ID NO-102).

FIG. 19 shows the alignment of the amino acid sequence of the heavychain variable region of 12B7 with the human germline V_(H) 1-69 aminoacid sequence (SEQ ID NO: 102).

FIG. 20 shows the alignment of the amino acid sequence of the heavychain variable region of 13G4 with the human germline V_(H) 3-9 aminoacid sequence (SEQ ID NO: 104).

FIG. 21 shows the alignment of the amino acid sequence of the lightchain variable region of 3G10 with the human germline V_(k) L6 aminoacid sequence (SEQ ID NO:105).

FIG. 22 shows the alignment of the amino acid sequence of the lightchain variable region of 12A4 with the human germline V_(k) L6 aminoacid sequence (SEQ ID NO:105).

FIG. 23 shows the alignment of the amino acid sequence of the lightchain variable region of 10A5 with the human germline V_(k) L15 aminoacid sequence (SEQ ID NO:106).

FIG. 24 shows the alignment of the amino acid sequence of the lightchain variable region of 5F8 with the human germline V_(k) A27 aminoacid sequence (SEQ ID NO: 107).

FIG. 25 shows the alignment of the amino acid sequence of the lightchain variable region of 10H10 with the human germline V_(k) L15 aminoacid sequence (SEQ ID NO:106).

FIG. 26 shows the alignment of the amino acid sequence of the lightchain variable region of 1B12 with the human germline V_(k) L6 aminoacid sequence (SEQ ID NO: 105).

FIG. 27 shows the alignment of the amino acid sequence of the lightchain variable region of 7H1 with the human germline V_(k) L6 amino acidsequence (SEQ ID NO:105).

FIG. 28 shows the alignment of the amino acid sequence of the lightchain variable region of 11E6 with the human germline V_(k) A27 aminoacid sequence (SEQ ID NO:107).

FIG. 29 shows the alignment of the amino acid sequence of the lightchain variable region of 11E6a (SEQ ID NO:109) with the human germlineV_(k) A27 amino acid sequence (SEQ ID NO:107).

FIG. 30 shows the alignment of the amino acid sequence of the lightchain variable region of 12B7 with the human germline V_(k) L6 aminoacid sequence (SEQ ID NO:105).

FIG. 31 shows the alignment of the amino acid sequence of the lightchain variable region of 13G4 with the human germline V_(k) L18 aminoacid sequence (SEQ ID NO:108).

FIGS. 32A-32C show the results of flow cytometry experimentsdemonstrating that the human monoclonal antibodies 3G10, 10A5, and 12A4,directed against human PD-L1, binds the cell surface of CHO cellstransfected with full-length human PD-L1. FIG. 32A is a Flow cytometryplot for 3G10, FIG. 32B is a Flow cytometry plot for 10A5, and FIG. 32Cis a Flow cytometry plot for 12A4.

FIG. 33 shows the results of flow cytometry experiments demonstratingthat the human monoclonal antibodies 3G10, 10A5, and 12A4, directedagainst human PD-L1, binds the cell surface of CHO cells transfectedwith full-length human PD-L1 in a concentration dependent manner.

FIG. 34 shows the results of ELISA experiments demonstrating that thehuman monoclonal antibodies 3G10, 10A5, and 12A4, directed against humanPD-L1, binds to PD-L1-Fc fusion protein.

FIG. 35 shows the results of experiments demonstrating HuMab titrationon stimulated human CD4+T cells.

FIG. 36 shows the results of experiments demonstrating HuMab titrationon stimulated cynomolgus PBMC.

FIGS. 37A-37C show the results of flow cytometry experimentsdemonstrating that the human monoclonal antibodies 3G10, 10A5, and 12A4,directed against human PD-L1, binds to PD-L1 on the cell surface ofactivated T cells. FIG. 37A is a Flow cytometry plot for 3G10, FIG. 37Bis a Flow cytometry plot for 10A5, and FIG. 37C is a Flow cytometry plotfor 12A4.

FIG. 38 demonstrates binding of HuMabs to ES-2 cells.

FIGS. 39A-39D show the results of experiments demonstrating that humanmonoclonal antibodies against human PD-L1 promote T-cell proliferation,IFN-γ secretion and IL-2 secretion in a mixed lymphocyte reaction assay.FIG. 39A is a bar graph showing concentration dependent T-cellproliferation using HuMAb 10A5; FIG. 39B is a bar graph showingconcentration dependent IFN-γ secretion using HuMAb 10A5; FIG. 39C is abar graph showing IFN-γ secretion using HuMAbs 3G10 and 12A4; FIG. 39Dis a bar graph showing concentration dependent IL-2 secretion usingHuMAb 10A5.

FIG. 40 demonstrates the effect of human anti-PD-L1 antibody onproliferation and IFN-γ secretion in the MLR using allogeneic dendriticcells and T cells (CD4+ effector T cells) Dendritic Cells.

FIGS. 41A and 41B show the results of experiments demonstrating thathuman monoclonal antibodies against human PD-L1 promote T-cellproliferation and IFN-γ secretion in MLR containing T regulatory cells.FIG. 41A is a bar graph showing concentration dependent T-cellproliferation using HuMAb 10A5; FIG. 41B is a bar graph showingconcentration dependent IFN-γ secretion using HuMAb 10A5.

FIG. 42 demonstrates the results of anti-PD-L1 antibodies on cellproliferation in a Mixed Lymphocyte Reaction in the presence ofregulatory T cells.

FIG. 43 demonstrates the results of anti-PD-L1 antibodies on cytokineproduction in a Mixed Lymphocyte Reaction in the presence of regulatoryT cells.

FIG. 44 demonstrates the results of anti-PD-L1 antibodies on CMV lysatestimulated human PBMC IFN-γ secretion.

FIG. 45 shows the results of flow cytometry experiments demonstratingthat human monoclonal antibodies against human PD-L1 block the bindingof PD-L1 to CHO transfected cells expressing PD-1.

FIG. 46 shows that anti-PD-L1 antibodies block binding of PD-1 to IFNγtreated ES-2 cells.

FIG. 47 shows the effect of anti-PD-L1 antibodies on tumor growth invivo.

DETAILED DESCRIPTION

In one aspect, the present disclosure relates to isolated monoclonalantibodies, particularly human monoclonal antibodies that bindspecifically to PD-L1. In certain embodiments, the antibodies of theinvention exhibit one or more desirable functional properties, such ashigh affinity binding to PD-L1, the ability to augment T cellproliferation, IFN-γ and/or IL-2 secretion in mixed lymphocytereactions, the ability to inhibit binding of PD-L1 to the PD-1 receptor,the ability to stimulate antibody responses and/or the ability toreverse the suppressive function of T regulatory cells. Additionally oralternatively, the antibodies of the invention are derived fromparticular heavy and light chain germline sequences and/or compriseparticular structural features such as CDR regions comprising particularamino acid sequences.

The instant disclosure provides, for example, isolated antibodies,methods of making such antibodies, immunoconjugates and bispecificmolecules comprising such antibodies and pharmaceutical compositionscontaining the antibodies, immunconjugates or bispecific molecules ofthe invention.

In another aspect, the disclosure pertains to methods of inhibitinggrowth of tumor cells in a subject using anti-PD-L1 antibodies. Theinvention also relates to methods of using the antibodies to modify animmune response, as well as to treat diseases such as cancer orinfectious disease, or to stimulate a protective autoimmune response orto stimulate antigen-specific immune responses (e.g., bycoadministration of anti-PD-L1 with an antigen of interest).

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present invention is the PD-L1 receptor.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: PR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., PD-L). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); ea e.g., Bird et al. (1988) Science 22:423-426; and Huston etal. (1988) Proc. Natl. Acad Set. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds PD-L1 is substantially free of antibodies that specifically bindantigens other than PD-L1). An isolated antibody that specifically bindsPD-L1 may, however, have cross-reactivity to other antigens, such asPD-L1 molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody,” as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody,” as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies awe sequences that, while derived from andrelated to human germline V_(n) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds to human PD-L1” isIntended to refer to an antibody that binds to human PD-L1 with a K_(D)of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, more preferably1×10⁻⁸ M or less, more preferably 5×10⁻⁹ M or less, even more preferablybetween 1×10⁻⁸ M and 1×10⁻¹⁰ M or less.

The term “K_(assoc)” or “K_(a),” as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D),” as, used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M orless, even more preferably 10⁻⁹ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

Anti-PD-L1 Antibodies

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to human PD-L1. Preferably, an antibody ofthe invention binds to PD-L1 with high affinity, for example with aK_(D) of 1×10⁻⁷ M or less. The anti-PD-L1 antibodies of the inventionpreferably exhibit one or more of the following characteristics:

-   -   (a) binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M or less;    -   (b) increases T-cell proliferation in a mixed lymphocyte        reaction (MLR) assay;    -   (c) increases interferon-γ production in an MLR assay;    -   (d) increases IL-2 secretion in an MLR assay    -   (e) stimulates antibody responses; and/or    -   (f) reverses the effect of T regulatory cells on T cell effector        cells and/or dendritic cells.

Preferably, the antibody binds to human PD-L1 with a K_(D) of 5×10⁻⁸ Mor less, binds to human PD-L1 with a K_(D) of 1×10⁻⁸ M or less, binds tohuman PD-L1 with a K_(D) of 5×10⁻⁹ M or less, binds to human PD-L1 witha K_(D) of 4×10⁻⁹ M or less, binds to human PD-L1 with a K_(D) of 2×10⁻⁹M or less, or binds to human PD-L1 with a K_(D) of between 1×10⁻⁹ M and1×10⁻¹⁰ M or less.

Standard assays to evaluate the binding ability of the antibodies towardPD-L1 are known in the art, including for example, ELISAs, Western blotsand RIAs. Suitable assays are described in detail in the Examples. Thebinding kinetics (e.g., binding affinity) of the antibodies also can beassessed by standard assays known in the art, such as by Biacore®analysis.

Monoclonal Antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 11E6, 12B7,and 13G4.

Preferred antibodies of the invention are the human monoclonalantibodies 30G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and13G4, isolated and structurally characterized as described in Examples 1and 2. The V_(H) amino acid sequences of 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7, and 13G4 are shown in SEQ ID NOs:1, 2, 3, 4, 5,6, 7, 8, 9, and 10, respectively. The V_(L) amino acid sequences of3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 are shownin SEQ ID NOs:11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, respectively.

Given that each of these antibodies can bind to PD-L1, the V_(H) andV_(L) sequences can be “mixed and matched” to create other anti-PD-L1binding molecules of the invention. PD-L1 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISAs). Preferably, when V_(H) andV_(L) chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence.

Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8,9, and 10; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:11, 12, 13, 14, 15, 16,17, 18, 19, and 20;

wherein the antibody specifically binds PD-L1, preferably human PD-L1.

Preferred heavy and light chain combinations include:

-   -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:1; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:11; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:2; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:12; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:3; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:13; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:4; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:14; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:5; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:15; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:6; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:16; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:7; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:17; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:8; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:18; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO:9; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:19; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 10; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO:20.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 3G10, 12A4, 10A5,5F, 8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, or combinations thereof.The amino acid sequences of the V_(H) CDR1s of 3G10, 12A4, 10A5, 5F81,100, 1B12, 7H1, 11E6, 12B7, and 13G4 are shown in SEQ ID NOs:21, 22, 23,24, 25, 26, 27, 28, 29, and 30, respectively. The amino acid sequencesof the V_(H) CDR2s of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11B6,12B7, and 13G4 are shown in SEQ ID NOs:31, 32, 33, 34, 35, 36, 37, 38,39, and 40, respectively. The amino acid sequences of the V_(H) CDR3s of3G1, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 are shownin SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, respectively.The amino acid sequences of the V_(k) CDR1s of 3G10, 12A4, 10A5, 5F8,10H0, 1B12, 7H11, 11E6, 12B7, and 13G4 are shown in SEQ ID NOs:51, 52,53, 54, 55, 56, 57, 58, 59, and 60, respectively. The amino acidsequences of the V_(k) CDR2s of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1,11E6, 12B7, and 13G4 are shown in SEQ ID NOs:61, 62, 63, 64, 65, 66, 67,68, 69, and 70, respectively. The amino acid sequences of the V_(k)CDR3s of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4are shown in SEQ ID NOs:71, 72, 73, 74, 75, 76, 77, 78, 79, and 80,respectively. The CDR regions are delineated using the Kabat system(Kabat, B. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242).

Given that each of these antibodies can bind to PD-L1 and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(k) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, CDR2, and CDR3 and a V_(k) CDR1, CDR2, and CDR3)to create other anti-PD-L1 binding molecules of the invention. PD-L1binding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., ELISAs,Biacore analysis). Preferably, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(k) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V sequence preferably is replacedwith a structurally similar CDR sequence(s). It will be readily apparentto the ordinarily skilled artisan that novel V_(H) and V_(L) sequencescan be created by substituting one or more V_(H) and/or V_(L) CDR regionsequences with structurally similar sequences from the CDR sequencesdisclosed herein for monoclonal antibodies antibodies 3G10, 12A4, 10A5,5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4.

Accordingly, in another aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:21, 22, 23, 24, 25, 26,27, 28, 29, and 30;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:31, 32, 33, 34, 35, 36,37, 38, 39, and 40;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:41, 42, 43, 44, 45, 46,47, 48, 49, and 50;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:51, 52, 53, 54, 55, 56,57, 58, 59, and 60;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:61, 62, 63, 64, 65, 66,67, 68, 69, and 70; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:71, 72, 73, 74, 75, 76,77, 78, 79, and 80;

wherein the antibody specifically binds PD-L1, preferably human PD-L1.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:21;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:31;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:41;

(d) a light chain variable region CDR1 comprising SEQ ID NO:51;

(c) a light chain variable region CDR2 comprising SEQ ID NO:61; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:71.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:22;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:32;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:42;

(d) a light chain variable region CDR1 comprising SEQ ID NO:52;

(e) a light chain variable region CDR2 comprising SEQ ID NO:62; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:72.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:23;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:33;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:43;

(d) a light chain variable region CDR1 comprising SEQ ID NO:53;

(e) a light chain variable region CDR2 comprising SEQ ID NO:63; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:73.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:24;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:34;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:44;

(d) a light chain variable region CDR1 comprising SEQ ID NO:54;

(e) a light chain variable region CDR2 comprising SEQ ID NO:64; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:74.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:25;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:35;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:45;

(d) a light chain variable region CDR1 comprising SEQ ID NO:55;

(e) a light chain variable region CDR2 comprising SEQ ID NO:65; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:75.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:26;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:36;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:46;

(d) a light chain variable region CDR1 comprising SEQ ID NO:56;

(e) a light chain variable region CDR2 comprising SEQ ID NO:66; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:76.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:27;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:37;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:47;

(d) a light chain variable region CDR1 comprising SEQ ID NO:57;

(e) a light chain variable region CDR2 comprising SEQ ID NO:67; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:77.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:28;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:38;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:48;

(d) a light chain variable region CDR1 comprising SEQ ID NO:58;

(e) a light chain variable region CDR2 comprising SEQ ID NO:68; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:78.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:29;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:39;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:49;

(d) a light chain variable region CDR1 comprising SEQ ID NO:59;

(e) a light chain variable region CDR2 comprising SEQ ID NO:69; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:79.

In another preferred embodiment the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO:30;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO:40;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO:50;

(d) a light chain variable region CDR1 comprising SEQ ID NO:60;

(e) a light chain variable region CDR2 comprising SEQ ID NO:70; and

(f) a light chain variable region CDR3 comprising SEQ ID NO:80.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, for example, Klimka et al., British J. ofCancer 83(2):252-260 (2000) (describing the production of a humanizedanti-CD30 antibody using only the heavy chain variable domain CDR3 ofmurine anti-CD30 antibody Ki-4); Beiboer at al., J. Mol. Biol.26:833-849 (2000) (describing recombinant epithelial glycoprotein-2(EGP-2) antibodies using only the heavy chain CDR3 sequence of theparental murine MOC-31 anti-EGP-2 antibody); Rader at al., Proc. Natl.Acad Sa. U.S.A. 9:8910-8915 (1998) (describing a panel of humanizedanti-integrin α_(v)β₃ antibodies using a heavy and light chain variableCDR3 domain of a murine anti-integrin α_(v)β₃ antibody LM609 whereineach member antibody comprises a distinct sequence outside the CDR3domain and capable of binding the same epitope as the parent muringantibody with affinities as high or higher than the parent murineantibody); Barbas at al., J. Am. Chem. Soc. 116:2161-2162 (1994)(disclosing that the CDR3 domain provides the most significantcontribution to antigen binding); Barbas at al., Proc. Natl. Acad. Sci.U.S.A. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3sequences of three Fabs (SI-1, SI-40, and SI-32) against human placentalDNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacingthe existing heavy chain CDR3 and demonstrating that the CDR3 domainalone conferred binding specificity); and Ditzel et al., J Immunol.157:739-749 (1996) (describing grafting studies wherein transfer of onlythe heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chainof a monospecific IgG tetanus toxoid-binding Fab p313 antibody wassufficient to retain binding specificity of the parent Fab). Each ofthese references is hereby incorporated by reference in its entirety.

Accordingly, within certain aspects, the present invention providesmonoclonal antibodies comprising one or more heavy and/or light chainCDR3 domain from a non-human antibody, such as a mouse or rat antibody,wherein the monoclonal antibody is capable of specifically binding toPD-L1. Within some embodiments, such inventive antibodies comprising oneor more heavy and/or light chain CDR3 domain from a non-human antibody(a) are capable of competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental non-human antibody.

Within other aspects, the present invention provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a first human antibody, such as, for example, a human antibodyobtained from a non-human animal, wherein the first human antibody iscapable of specifically binding to PD-L1 and wherein the CDR3 domainfrom the first human antibody replaces a CDR3 domain in a human antibodythat is lacking binding specificity for PD-L1 to generate a second humanantibody that is capable of specifically binding to PD-L1. Within someembodiments, antibodies of the instant disclosure comprising one or moreheavy and/or light chain CDR3 domain from the first human antibody (a)are capable of competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental first human antibody.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereofcomprising a heavy chain variable region that is the product of orderived from a human V_(H) 1-18 gene, wherein the antibody specificallybinds PD-L1. In another preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereofcomprising a heavy chain variable region that is the product of orderived from a human V_(H) 1-69 gene, wherein the antibody specificallybinds PD-L1. In another preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereofcomprising a heavy chain variable region that is the product of orderived from a human V_(H) 1-3 gene, wherein the antibody specificallybinds PD-L1. In another preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 3-9 gene, wherein the antibody specificallybinds PD-L1. In yet another preferred embodiment, the invention providesan isolated monoclonal antibody, or an antigen-binding portion thereof,comprising a light chain variable region that is the product of orderived from a human V_(K) L6 gene, wherein the antibody specificallybinds PD-L1. In yet another preferred embodiment, the invention providesan isolated monoclonal antibody, or an antigen-binding portion thereof,comprising a light chain variable region that is the product of orderived from a human V_(K) L15 gene, wherein the antibody specificallybinds PD-L1. In yet another preferred embodiment, the invention providesan isolated monoclonal antibody, or an antigen-binding portion thereofcomprising a light chain variable region that is the product of orderived from a human V_(K) A27 gene, wherein the antibody specificallybinds PD-L1. In yet another preferred embodiment, the invention providesan isolated monoclonal antibody, or an antigen-binding portion thereofcomprising a light chain variable region that is the product of orderived from a human V_(K) L18 gene, wherein the antibody specificallybinds PD-L1. In yet another preferred embodiment, the invention providesan isolated monoclonal antibody, or antigen-binding portion thereof,wherein the antibody:

(a) comprises a heavy chain variable region that is the product of orderived from a human V_(H) 1-18, 1-69, 1-3 or 3-9 gene (which encodesthe amino acid sequences set forth in SEQ ID NOs:101, 102, 103 and 104,respectively);

(b) comprises a light chain variable region that is the product of orderived from a human V_(K) L6, L15, A27 or L18 gene (which encodes theamino acid sequences set forth in SEQ ID NOs:105, 106, 107 and 108,respectively); and

(c) specifically binds to PD-L1, preferably human PD-L1.

An example of an antibody having V_(H) and V_(K) of V_(H) 1-18 and V_(K)L6, respectively, is 3G10. Examples of antibodies having V_(H) and V_(K)of V_(H) 1-69 and V_(K) L6, respectively, 12A4, 1B12, 7H1, and 12B7. Anexample of an antibody having V_(H) and V_(K) of V_(H) 1-3 and V_(K)L15, respectively, is 10A5. Examples of antibodies having V_(H) andV_(K) of V_(H) 1-69 and V_(K) A27, respectively, are 5F8, 11E6 and11B6a. An example of an antibody having V_(R) and V_(K) of V_(H) 3-9 andV_(K) L15, respectively, is 10H10. An example of an antibody havingV_(H) and V_(K) of V_(H) 1-3 and V_(K) L15, respectively, is 10A5. Anexample of an antibody having V_(H) and V_(K) of V_(H) 3-9 and V_(K)L18, respectively, is 13G4.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (I.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody is generally at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. In certain embodiments, a human antibody derivedfrom a particular human germline sequence will display no more than 10amino acid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain other embodiments, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gone.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-PD-L1 antibodies of the invention.

For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof comprising a heavy chain variable regionand a light chain variable region, wherein:

-   -   (a) the heavy chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs:1, 2,        3, 4, 5, 6, 7, 8, 9, and 10;    -   (b) the light chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs:11,        12, 13, 14, 15, 16, 17, 18, 19, and 20;    -   (c) the antibody binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M        or less;    -   (d) the antibody increases T-cell proliferation in a mixed        lymphocyte reaction (MLR) assay;    -   (e) the antibody increases interferon-γ production in an MLR        assay;    -   (f) the antibody increases IL-2 secretion in an MLR assay,    -   (g) the antibody stimulates antibody responses; and    -   (h) reverses the effect of T regulatory cells on T cell effector        cells and/or dendritic cells.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85% 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences set forthabove. An antibody having V_(H) and V_(L) regions having high (i.e., 80%or greater) homology to the V_(H) and V_(L) regions of the sequences setforth above, can be obtained by mutagenesis (e.g., site-directed orPCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ IDNOs:25, 26, 27, 28, 29, and 30, followed by testing of the encodedaltered antibody for retained function (I.e., the functions set forth in(c) through (h) above) using the functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

In certain instances, the protein sequences of the present disclosurecan be further used as a “query sequence” to perform a search againstpublic databases to, for example, identify related sequences. Suchsearches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 21(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4), orconservative modifications thereof, and wherein the antibodies retainthe desired functional properties of the anti-PD-L1 antibodies of theinvention. Accordingly, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof; comprising a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences,wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, andconservative modifications thereof;

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs:71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, andconservative modifications thereof,

(c) the antibody binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M or less;

(d) the antibody increases T-cell proliferation in a mixed lymphocytereaction (MLR) assay;

(e) the antibody increases interferon-γ production in an MLR assay;

(f) the antibody increases IL-2 secretion in an MLR assay

(g) the antibody stimulates antibody responses; and

(h) reverses the effect of T regulatory cells on T cell effector cellsand/or dendritic cells.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs:31, 32, 33, 34, 35, 36, 37, 38, 39,and 40, and conservative modifications thereof; and the light chainvariable region CDR2 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs:61, 62,63, 64, 65, 66, 67, 68, 69, and 70, and conservative modificationsthereof. In another preferred embodiment, the heavy chain variableregion CDR1 sequence comprises an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOs:21, 22, 23, 24,25, 26, 27, 28, 29, and 30, and conservative modifications thereof; andthe light chain variable region CDR1 sequence comprises an amino acidsequence selected from the group consisting of amino acid sequences ofSEQ ID NOs:51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, and conservativemodifications thereof.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth in (c) through (h) above) usingthe functional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-PD-L1 Antibodies of theInvention

In another embodiment, the invention provides antibodies that bind tothe same epitope on human PD-L1 as any of the PD-L1 monoclonalantibodies of the invention (i.e., antibodies that have the ability tocross-compete for binding to PD-L1 with any of the monoclonal antibodiesof the invention). In preferred embodiments, the reference antibody forcross-competition studies can be the monoclonal antibody 3G10 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs:1 and 11,respectively), or the monoclonal antibody 12A4 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs:2 and 12, respectively), or themonoclonal antibody 10A5 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs:3 and 13, respectively), or the monoclonal antibody 10A5(having V_(H) and V_(L) sequences as shown in SEQ ID NOs:3 and 13,respectively), or the monoclonal antibody 5F8 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs:4 and 14, respectively), or themonoclonal antibody 10H10 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs:5 and 15, respectively), or the monoclonal antibody 1B12(having V_(H) and V_(L) sequences as shown in SEQ ID NOs:6 and 16,respectively), or the monoclonal antibody 7H1 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs:7 and 17, respectively), or themonoclonal antibody 11E6 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs:8 and 18, respectively), or the monoclonal antibody 12B7(having V_(H) and V_(L) sequences as shown in SEQ ID NOs:9 and 19,respectively), or the monoclonal antibody 13G4 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs:10 and 20, respectively). Suchcross-competing antibodies can be identified based on their ability tocross-compete with 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7or 13G4 in standard PD-L1, binding assays. For example, BIAcoreanalysis, ELISA assays or flow cytometry may be used to demonstratecross-competition with the antibodies of the current invention. Theability of a test antibody to inhibit the binding of, for example, 3G10,12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4, to human PD-L1demonstrates that the test antibody can compete with 3G10, 12A4, 10A5,5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 for binding to human PD-L1 andthus binds to the same epitope on human PD-L1 as 3G10, 12A4, 10A8,10H10, 1B12, 7H1, 11E6, 12B7 or 13G4. In a preferred embodiment, theantibody that binds to the same epitope on human PD-L1 as 3G10, 12A4,10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 is a human monoclonalantibody. Such human monoclonal antibodies can be prepared and isolatedas described in the Examples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature32:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al.(1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.) Accordingly, another embodiment of theinvention pertains to an isolated monoclonal antibody, or antigenbinding portion thereof, comprising a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 sequences comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:21, 22, 23,24, 25, 26, 27, 28, 29, and 30, SEQ ID NOs:31, 32, 33, 34, 35, 36, 37,38, 39, and 40, and SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, and50, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 sequences comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:51, 52, 53, 54, 55, 56, 57, 58, 59,and 60, SEQ ID NOs:61, 62, 63, 64, 65, 66, 67, 68, 69, and 70, and SEQID NOs:71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, respectively. Thus,such antibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4yet may contain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A, et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M, et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of VS Segments with Different Hypervariable Loops” J. Mol.Biol. 227:776-798; and Cox, J. P. L et al. (1994) “A Directory of HumanGerm-line V_(H) Segments Reveals a Strong Bias in their Usage” Eur. J.Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research2:3389-3402), which is well known to those skilled in the art. BLAST isa heuristic algorithm in that a statistically significant alignmentbetween the antibody sequence and the database sequence is likely tocontain high-scoring segment pairs (HSP) of aligned words. Segment pairswhose scores cannot be improved by extension or trimming is called ahit. Briefly, the nucleotide sequences of VBASE origin(vbase.mrc-cpe.cam.ac.uk/vbase/list2.php) are translated and the regionbetween and including FR1 through FR3 framework region is retained. Thedatabase sequences have an average length of 98 residues. Duplicatesequences which are exact matches over the entire length of the proteinare removed. A BLAST search for proteins using the program blastp withdefault, standard parameters except the low complexity filter which isturned off and the substitution matrix of BLOSUM62, filters for top 5hits yielding sequence matches. The nucleotide sequences are translatedin all six frames and the frame with no stop codons in the matchingsegment of the database sequence is considered the potential hit. Thisis in turn confirmed using the BLAST program tblastx. This translatesthe antibody sequence in all six frames and compares those translationsto the VBASE nucleotide sequences dynamically translated in all sixframes.

The identities are exact amino acid matches between the antibodysequence and the protein database over the entire length of thesequence. The positives (identities+ substitution match) are notidentical but amino acid substitutions guided by the BLOSUM62substitution matrix. If the antibody sequence matches two of thedatabase sequences with same identity, the hit with most positives wouldbe decided to be the matching sequence hit.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the V_(H) 1-18framework sequences (SEQ ID NO:101) and/or the V_(H) 1-69 frameworksequences (SEQ ID NO: 102) and/or the V_(H) 1-3 framework sequences (SEQID NO:103) and/or the V_(H) 3-9 framework sequences (SEQ ID NO: 104)and/or the V_(K) L6 framework sequences (SEQ ID NO:105) and/or the V_(K)L15 framework sequences (SEQ ID NO:106) and/or the V_(K) A27 frameworksequences (SEQ ID NO:107) and/or the V_(K) L18 framework sequences (SEQID NO: 107) used by preferred monoclonal antibodies of the invention.The V CDR1, CDR2, and CDR3 sequences, and the V_(K) CDR1, CDR2, and CDR3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(K) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the invention provides isolatedanti-PD-L1 monoclonal antibodies, or antigen binding portions thereofcomprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs:21, 22,23, 24, 25, 26, 27, 28, 29, and 30; (b) a V_(H) CDR2 region comprisingan amino acid sequence selected from the group consisting of SEQ IDNOs:31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs:31, 32, 33, 34, 35, 36,37, 38, 39, and 40; (c) a V_(H) CDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:41, 42, 43,44, 45, 46, 47, 48, 49, and 50, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49,and 50; (d) a V_(K) CDR1 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:51, 52, 53, 54, 55, 56,57, 58, 59, and 60, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs:51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; (e) aV_(K) CDR2 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:61, 62, 63, 64, 65, 66, 67, 68, 69, and70, or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NOs:61,62, 63, 64, 65, 66, 67, 68, 69, and 70; and (f) a V_(K) CDR3 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs:71, 72, 73, 74, 75, 76,77, 78, 79, and 80.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(K), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. For example, as described below, anumber of amino acid changes in the framework regions of the anti-PD-L1antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4that differ from the parent germline sequence. To return the frameworkregion sequences to their germline configuration, the somatic mutationscan be “backmutated” to the germline sequence by, for example,site-directed mutagenesis or PCR-mediated mutagenesis. The alignment ofthe V_(H) region for 3G10 against the parent germline V_(H) 1-18sequence is shown in FIG. 11. The alignment of the V_(H) region for 12A4against the parent germline V_(H) 1-69 sequence is shown in FIG. 12. Thealignment of the V_(H) region for 10A5 against the parent germline V_(H)1-3 sequence is shown in FIG. 13. The alignment of the V_(H) region for5F8 against the parent germline V_(H) 1-69 sequence is shown in FIG. 14.The alignment of the V_(H) region for 10H10 against the parent germlineV_(H) 3-9 sequence is shown in FIG. 15. The alignment of the V_(H)region for 1B12 against the parent germline V_(H) 1-69 sequence is shownin FIG. 16. The alignment of the V_(a) region for 7H1 against the parentgermline V_(H) 1-69 sequence is shown in FIG. 17. The alignment of theV_(H) region for 11E6 against the parent germline V_(H) 1-69 sequence isshown in FIG. 18. The alignment of the Via region for 12B7 against theparent germline V_(H) 1-69 sequence is shown in FIG. 19. The alignmentof the V_(H) region for 13G4 against the parent germline V_(H) 3-9sequence is shown in FIG. 20.

For example, for 3G10, amino acid residue #79 (within FR3) of V_(H) is avaline whereas this residue in the corresponding V_(H) 1-18 germlinesequence is an alanine. To return the framework region sequences totheir germline configuration, the somatic mutations can be “backmutated”to the germline sequence by, for example, site-directed mutagenesis orPCR-mediated mutagenesis (e.g., residue #79 (residue #13 of FR3) of theV_(H) of 3G10 can be “backmutated” from valine to alanine).

As another example, for 12A4, amino acid residue #24 (within FR1) ofV_(H) is a threonine whereas this residue in the corresponding V_(H)1-69 germline sequence is an alanine. To return the framework regionsequences to their germline configuration, for example, residue #24 ofthe V_(H) of 12A4 can be “backmutated” from threonine to alanine. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

As another example, for 12A4, amino acid residue #27 (within FR1) ofV_(H) is an aspartic acid whereas this residue in the correspondingV_(H) 1-69 germline sequence is a glycine. To return the frameworkregion sequences to their germline configuration, for example, residue#27 of the V_(H) of 12A4 can be “backmutated” from aspartic acid toglycine. Such “backmutated” antibodies are also intended to beencompassed by the invention.

As another example, for 12A4, amino acid residue #95 (within FR3) ofV_(H) is a phenylalanine whereas this residue in the corresponding V_(H)1-69 germline sequence is a tyrosine. To return the framework regionsequences to their germline configuration, for example, residue #95(residue #29 of PR3) of the V_(H) of 12A4 can be “backmutated” fromphenylalanine to tyrosine. Such “backmutated” antibodies are alsointended to be encompassed by the invention.

As another example, for 5F8, amino acid residue #24 (within FR1) is avaline whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an alanine. To return the framework region sequences totheir germline configuration, for example, residue #24 of the V_(H) of5F8 can be “backmutated” from valine to alanine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 5F8, amino acid residue #28 (within FR1) is anisoleucine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an threonine. To return the framework region sequences totheir germline configuration, for example, residue #28 of the V_(H) of5F8 can be “backmutated” from isoleucine to threonine. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

As another example, for 10H10, amino acid residue #24 (within FR1) is avaline whereas this residue in the corresponding V_(H) 3-9 germlinesequence is an alanine. To return the framework region sequences totheir germline configuration, for example, residue #24 of the V_(H) of10H10 can be “backmutated” from valine to alanine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 10H10, an amino acid can be inserted followingamino acid residue #97 (within FR3). This amino acid is a valine. Toreturn the framework region sequences to their germline configuration,for example, the inserted amino acid following residue #97 of the V_(H)of 10H10 can be “backmutated” to delete this valine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 1B12, amino acid residue #24 (within FR1) is athreonine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an alanine. To return the framework region sequences totheir germline configuration, for example, residue #24 of the V_(H) ofB12 can be “backmutated” from threonine to alanine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 1B12, amino acid residue #27 (within FR1) is anaspartic acid whereas this residue in the corresponding V_(H) 1-69germline sequence is an glycine. To return the framework regionsequences to their germline configuration, for example, residue #27 ofthe V_(H) of 1B12 can be “backmutated” from aspartic acid to glycine.Such “backmutated” antibodies are also intended to be encompassed by theinvention.

As another example, for 1B12, amino acid residue #95 (within FR3) is aphenylalanine whereas this residue in the corresponding V_(H) 1-69germline sequence is an tyrosine. To return the framework regionsequences to their germline configuration, for example, residue #95(residue #29 of FR3) of the V_(H) of 1B12 can be “backmutated” fromphenylalanine to tyrosine. Such “backmutated” antibodies are alsointended to be encompassed by the invention.

As another example, for 7H1, amino acid residue #24 (within FR1) is athreonine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an alanine. To return the framework region sequences totheir germline configuration, for example, residue #24 of the V_(H) of7H1 can be “backmutated” from threonine to alanine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 7H1, amino acid residue #77 (within FR3) is athreonine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is a serine. To return the framework region sequences to theirgermline configuration, for example, residue #72 (residue #11 of FR3) ofthe V_(H) of 7H1 can be “backmutated” from threonine to serine. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

As another example, for 11E6, amino acid residue #78 (within FR3) is analanine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is a threonine. To return the framework region sequences totheir germline configuration, for example, residue #78 (residue 12 ofFR3) of the V_(H) of 11E6 can be “backmutated” from alanine tothreonine. Such “backmutated” antibodies are also intended to beencompassed by the invention.

As another example, for 12B7, amino acid residue #13 (within FR1) is aglutamic acid whereas this residue in the corresponding V_(H) 1-69germline sequence is an lysine. To return the framework region sequencesto their germline configuration, for example, residue #13 of the V_(H)of 12B7 can be “backmutated” glutamic acid to lysine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 12B7, amino acid residue #30 (within FR1) is anasparagine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an serine. To return the framework region sequences to theirgermline configuration, for example, residue #30 of the V_(H) of 12B7can be “backmutated” from asparagine to serine. Such “backmutated”antibodies are also intended to be encompassed by the invention.

As another example, for 12B7, amino acid residue #77 (within FR3) is anasparagine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an serine. To return the framework region sequences to theirgermline configuration, for example, residue #377 (residue 11 of FR3) ofthe V_(H) of 12B7 can be “backmutated” from asparagine to serine. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

As another example, for 12B7, amino acid residue #82 (within FR3) is anaspartic acid whereas this residue in the corresponding V_(H) 1-69germline sequence is a glutamic acid. To return the framework regionsequences to their germline configuration, for example, residue #82(residue #16 of FR3) of the V_(H) of 1217 can be “backmutated” fromaspartic acid to glutamic acid. Such “backmutated” antibodies are alsointended to be encompassed by the invention.

As another example, for 13G4, amino acid residue #27 (within FR1) Is anisoleucine whereas this residue in the corresponding V_(H) 1-69 germlinesequence is an phenylalanine. To return the framework region sequencesto their germline configuration, for example, residue #27 of the V_(H)of 12B7 can be “backmutated” from isoleucine to phenylalanine. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Can et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of he antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcal protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta at al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fe region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fey receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.27:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/B333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

In certain other embodiments, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705,and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705, and Ms709 FUT8^(−/−) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 20040110704 by Yamane et al.and Yamano-Ohnuki et al. (2004) Biotechnol Bioeng 18:614-22). As anotherexample, EP 1,176,195 by Hanal et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. Hanal et al. also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFe region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J Biol. Chem.2:26733-26740). PCT Publication WO 99/54342 by Umana et al. describescell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III(GnTIII)) such that antibodies expressed in the engineered cell linesexhibit increased bisecting GlcNac structures which results in increasedADCC activity of the antibodies (see also Umana et al. (1999) Nat.Biotech. 12:176-180). Alternatively, the fucose residues of the antibodymay be cleaved off using a fucosidase enzyme. For example, thefucosidase alpha-L-fucosidase removes fucosyl residues from antibodies(Tarentino, A. L. et al. (1975) Biochem. 1:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof;typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

As discussed above, the anti-PD-L1 antibodies having V_(H) and V_(K)sequences disclosed herein can be used to create new anti-PD-L1antibodies by modifying the V_(H) and/or V_(K) sequences, or theconstant region(s) attached thereto. Thus, in another aspect of theinvention, the structural features of an anti-PD-L1 antibody of theinvention, e.g. 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or13G4, are used to create structurally related anti-PD-L1 antibodies thatretain at least one functional property of the antibodies of theinvention, such as binding to human PD-L1. For example, one or more CDRregions of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 71, 11E6, 12B7, or 13G4or mutations thereof; can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, anti-PD-L1 antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(K) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(K) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-PD-L1 antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs:21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, a CDR2 sequence selectedfrom the group consisting of SEQ ID NOs:31, 32, 33, 34, 35, 36, 37, 38,39, and 40, and/or a CDR3 sequence selected from the group consisting ofSEQ ID NOs:41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; and/or (i) alight chain variable region antibody sequence comprising a CDR1 sequenceselected from the group consisting of SEQ ID NOs:51, 52, 53, 54, 55, 56,57, 58, 59, and 60, a CDR2 sequence selected from the group consistingof SEQ ID NOs:61, 62, 63, 64, 65, 66, 67, 68, 69, and 70, and/or a CDR3sequence selected from the group consisting of SEQ ID NOs:71, 72, 73,74, 75, 76, 77, 78, 79, and 80;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-PD-L1 antibodies described herein, which functional propertiesinclude, but are not limited to:

-   -   (i) binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M or less;    -   (ii) increases T-cell proliferation in a mixed lymphocyte        reaction (MLR) assay;    -   (iii) increases interferon-γ production in an MLR assay;    -   (iv) increases IL-2 secretion in an MLR assay;    -   (v) stimulates antibody responses; and/or    -   (vi) reverses the effect of T regulatory cells on T cell        effector cells and/or dendritic cells.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., flow cytometry, bindingassays).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-PD-L1 antibody coding sequence and the resultingmodified anti-PD-L1 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Disclosure

Another aspect of the disclosure pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “readeredsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, for example, DNA or RNA and may ormay not contain intronic sequences. In a preferred embodiment; thenucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules of the invention are those encodingthe VH and VL sequences of the 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1,11E6, 12B7, and 13G4, monoclonal antibodies. DNA sequences encoding theVH sequences of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and13G4, are shown in SEQ ID NOs:81, 82, 83, 84, 85, 86, 87, 88, 89 and 90,respectively. DNA sequences encoding the VL sequences of 3G10, 12A4,10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, are shown in SEQ IDNOs:91, 92, 93, 94, 95, 96, 97, 98, 99 and 100, respectively.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked,” as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 0.5:5879-5883; McCafferty et al., (1990) Nature 48:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be produced bya variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.).

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstPD-L1 can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM Mice™, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 38(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann N.Y. Acad Sci. 764:536-546). The preparation anduse of HuMab mice, and the genomic modifications carried by such mice,is further described in Taylor, L. et al. (1992) Nucleic Acids Research20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656;Tuaillon et al. (1993) Proc. Natl. Acad. Sc. USA 90:3720-3724; Choi etal. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) 1 Immunol. 152:2912-2920; Taylor, L etal. (1994) International Immunology 6:579-591; and Fishwild, D. et al.(1996) Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM Mice™,” are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-PD-L1 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucheriapati et a.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-PD-L1 antibodies of the invention. For example, mice carrying botha human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka at al. (2000) Proc Natl. Acad Sc. USA 22:722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (Kuroiwa et al. (2002) NatureBiotechnology 20:889-894) and can be used to raise anti-PD-L1 antibodiesof the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of the invention,such mice can be immunized with a purified or enriched preparation ofPD-L1 antigen and/or recombinant PD-L1, or an PD-L1 fusion protein, asdescribed by Lonberg, N. et al. (1994) Nature 0.8(6474): 856-859;Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; and PCTPublication WO 98/24884 and WO 01/14424. Preferably, the mice will be6-16 weeks of age upon the first infusion. For example, a purified orrecombinant preparation (5-50 μg) of PD-L1 antigen can be used toimmunize the human Ig mice intraperitoneally.

Detailed procedures to generate fully human monoclonal antibodies toPD-L1 are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially Immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-PD-L1 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo12). Alternatively or additionally, the KMMouse™ strain can be used, as described in Example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theDisclosure

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×10⁵in flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe re-plated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80′ C.

Generation of Transfectomas Producing Monoclonal Antibodies of theDisclosure

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof;DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(K) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et a.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperty folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be Ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (Includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.USA 77:4216-4220, used with a DHFR selectable marker, e.g., as describedin R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSOmyeloma cells, COS cells and SP2 cells. In particular, for use with NSOmyeloma cells, another preferred expression system is the GS geneexpression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody Into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of the invention can be tested for binding to PD-1 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified PD-L1 at 0.25 μg/ml in PBS, and then blocked with 5% bovineserum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasmafrom PD-L1-immunized mice) are added to each well and incubated for 1-2hours at 37° C. The plates are washed with PBS/Tween and then incubatedwith secondary reagent (e.g., for human antibodies, a goat-anti-humanIgG Fc-specific polyclonal reagent) conjugated to alkaline phosphatasefor 1 hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, micewhich develop the highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with PD-L1 immunogen.Hybridomas that bind with high avidity to PD-L1 are subcloned andfurther characterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-PD-L1 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supenatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD1)using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-PD-L1 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using PD-L1 coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4′ C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-cojugated probes. Plates aredeveloped and analyzed as described above.

Anti-PD-L1 human IgGs can be further tested for reactivity with PD-L1antigen by Western blotting. Briefly, PD-L1 can be prepared andsubjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.After electophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Antibody Physical Properties

The antibodies of the present invention may be further characterized bythe various physical properties of the anti-PD-1, antibodies. Variousassays may be used to detect and/or differentiate different classes ofantibodies based on these physical properties.

In some embodiments, antibodies of the present invention may contain oneor more glycosylation sites in either the light or heavy chain variableregion. The presence of one or more glycosylation sites in the variableregion may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Ann Rev Biochem 41:673-702; Gala F A and MorrisonSL (2004) J Immunol 12:5489-94; Wallick et al. (1988) J Exp Med0.168:1099-109; Spiro R G (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 2:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. Variable region glycosylation may be tested using a Glycoblotassay, which cleaves the antibody to produce a Fab, and then tests forglycosylation using an assay that measures periodate oxidation andSchiff base formation. Alternatively, variable region glycosylation maybe tested using Dionex light chromatography (Dionex-LC), which cleavessaccharides from a Fab into monosaccharides and analyzes the individualsaccharide content. In some instances, it is preferred to have ananti-PD-L1 antibody that does not contain variable region glycosylation.This can be achieved either by selecting antibodies that do not containthe glycosylation motif in the variable region or by mutating residueswithin the glycosylation motif using standard techniques well known inthe art.

In a preferred embodiment, the antibodies of the present invention donot contain asparagine isomerism sites. A deamidation or isoasparticacid effect may occur on N-G or D-G sequences, respectively. Thedeamidation or isoaspartic acid effect results in the creation ofisoaspartic acid which decreases the stability of an antibody bycreating a kinked structure off a side chain carboxy terminus ratherthan the main chain. The creation of isoaspartic acid can be measuredusing an iso-quant assay, which uses a reverse-phase HPLC to test forisoaspartic acid.

Each antibody will have a unique isoelectric point (pI), but generallyantibodies will fall in the pH range of between 6 and 9.5. The pI for anIgG1 antibody typically falls within the pH range of 7-9.5 and the pIfor an IgG4 antibody typically falls within the pH range of 6-8.Antibodies may have a pI that is outside this range. Although theeffects are generally unknown, there is speculation that antibodies witha pI outside the normal range may have some unfolding and instabilityunder in vivo conditions. The isoelectric point may be tested using acapillary isoelectric focusing assay, which creates a pH gradient andmay utilize laser focusing for increased accuracy (Janini et a (2002)Electrophores 2: 1605-11; Ma et al. (2001) Chmronatographia 5:S75-89;Hunt et al (1998) J Chromatogr A 800:355-67). In some instances, it Ispreferred to have an anti-PD-L1 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range, or by mutating charged surfaceresidues using standard techniques well known in the art.

Each antibody will have a melting temperature that is indicative ofthermal stability (Krishnamurthy R and Manning M C (2002) Curr PharmBiotechnol 3:361-71). A higher thermal stability indicates greateroverall antibody stability in vivo. The melting point of an antibody maybe measure using techniques such as differential scanning calorimetry(Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) ImmunolLeu 68:47-52). T_(M1) indicates the temperature of the initial unfoldingof the antibody. T_(M2) indicates the temperature of complete unfoldingof the antibody. Generally, it is preferred that the T_(M1) of anantibody of the present invention is greater than 60° C., preferablygreater than 65′C, even more preferably greater than 70° C.Alternatively, the thermal stability of an antibody may be measure usingcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).The thermal stability of anti-PD-L1 antibodies disolosed herein issummarized in Table 1.

TABLE 1 Tm1 Tm2 mAb (° C.) (° C.) 3G10 70 75 5F8 72 74 11E6 64 73 1B1269 72 12A4 68 72 10A5 71 12B7 70 13G4 66 69 10H10 69

In a preferred embodiment, antibodies are selected that do not rapidlydegrade. Fragmentation of an anti-PD-L1 antibody may be measured usingcapillary elctrophoresis (CB) and MALDI-MS, as is well understood in theart (Alexander A J and Hughes D E (1995) Anal Chem 673626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects. Aggregation may lead to triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation may be measured by several techniques well known in the art,including size-exclusion column (SEC) high performance liquidchromatography (HPLC), and light scattering to identify monomers,dimers, trimers or multimers.

Immunoconjugates

In another aspect, the present invention features an anti-PD-L1antibody, or a fragment thereof; conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (H) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate Is commercially available(Mylotarg®; Wyeth-Ayerst).

Cytotoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat Rev. Cancer 2:750-763; Pastan, L andKreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter,P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunconjugates areestablished in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof; such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”) interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“C-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies Forimmunotargeting Of Drugs In Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy, Reisfold et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review.” in Monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe at al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,”Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-PD-L1 antibody, or a fragment thereon of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for PD-L1 and a secondbinding specificity for a second target epitope. In a particularembodiment of the invention, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, the invention includes bispecific molecules capable ofbinding both to FcγR or FcαR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to target cellsexpressing PD-L. These bispecific molecules target PD-L1 expressingcells to effector call and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of an PD-L1 expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

In an embodiment of the invention in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fe binding specificity and ananti-PD-L1 binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the F_(C) receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fe receptoror a target cell antigen. Alternatively, the anti-enhancement factorportion can bind to an entity that is different from the entity to whichthe first and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g. viaCD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et a. U.S. Pat. No. 4,946,778, the contents of whichis expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcγRI(CD64), FcγRI(CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹ M⁻¹).

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFcγ binding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immuno148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of the invention because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-PD-L1 binding specificities, using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad Sci. USA 82:8648). Other methodsinclude those described in Paulus (1985) Behring Ins. Mitt. No. 78,118-132; Brennan et al. (1985) Science 22:81-83), and Glennie et al.(1987) Immunol. 132: 2367-2375). Preferred conjugating agents are SATAand sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbant assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B, Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-PD-L1 antibody of the presentinvention combined with at least one other anti-inflammatory orimmunosuppressant agent. Examples of therapeutic agents that can be usedin combination therapy are described in greater detail below in thesection on uses of the antibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e. antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated Into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred per cent, this amount will range from about 0.01 per centto about ninety-nine percent of active ingredient, preferably from about0.1 per cent to about 70 per cent, most preferably from about 1 per centto about 30 per cent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-PD-L1antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patientin some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient in general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-PD-L1 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of PD-L1+tumors, a“therapeutically effective dosage” preferably inhibits cell growth ortumor growth by at least about 20, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to inhibit tumor growth can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 22:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. O. Bloeman et al. (1995) FEBS Lett. 35:140; M.Owais et al. (1995) Antimicrob. Agents Chemother. 3:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);p120 (Schreier et al. (1994) J Biol. Chem. 2:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett 46:123; J. J. Killion; I. J.Fidler (1994) Immunomethods 4:273.

Uses and Methods of the invention

The antibodies, antibody compositions and methods of the presentinvention have numerous in vitro and in vivo utilities involving, forexample, detection of PD-L1 or enhancement of immune response byblockade of PD-L1. In a preferred embodiment, the antibodies of thepresent invention are human antibodies. For example, these molecules canbe administered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to enhance immunity in a variety of situations.Accordingly, in one aspect, the invention provides a method of modifyingan immune response in a subject comprising administering to the subjectthe antibody, or antigen-binding portion thereof of the invention suchthat the immune response in the subject Is modified. Preferably, theresponse is enhanced, stimulated or up-regulated.

As used herein, the term “subject” is intended to include human andnon-human animals. Non-human animals includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cats,cows, horses, chickens, amphibians, and reptiles, although mammals arepreferred, such as non-human primates, sheep, dogs, cats, cows andhorses. Preferred subjects include human patients in need of enhancementof an immune response. The methods are particularly suitable fortreating human patients having a disorder that can be treated byaugmenting the T-cell mediated immune response. In a particularembodiment, the methods are particularly suitable for treatment ofcancer cells in vive. To achieve antigen-specific enhancement ofimmunity, the anti-PD-L1 antibodies can be administered together with anantigen of interest. When antibodies to PD-L1 are administered togetherwith another agent, the two can be administered in either order orsimultaneously.

The invention further provides methods for detecting the presence ofhuman PD-L1 antigen in a sample, or measuring the amount of human PD-L1antigen, comprising contacting the sample, and a control sample, with ahuman monoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to human PD-L1, under conditions that allow forformation of a complex between the antibody or portion thereof and humanPD-L1. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of human PD-L1 antigen in the sample.

Cancer

Blockade of PD-L1 by antibodies can enhance the immune response tocancerous cells in the patient. PD-L1 is not expressed in normal humancells, but is abundant in a variety of human cancers (Dong et al. (2002)Nat Med 8:787-9). The interaction between PD-1 and PD-L1 results in adecrease in tumor infiltrating lymphocytes, a decrease in T-cellreceptor mediated proliferation, and immune evasion by the cancerouscells (Dong et al. (2003) J Mol Med 81:281-7; Blank et al. (2004) CancerImmunol. Immunother. [epub]; Konishi et al. (2004) Clin. Cancer Res.10:5094-100). Immune suppression can be reversed by inhibiting the localinteraction of PD-L1 to PD-1 and the effect is additive when theinteraction of PD-L2 to PD-1 is blocked as well (Iwai et al. (2002) PNAS92:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66). An anti-PD-L1antibody may be used alone to inhibit the growth of cancerous tumors.Alternatively, an anti-PD-L1 antibody may be used in conjunction withother immunogenic agents, standard cancer treatments, or otherantibodies, as described below.

Accordingly, in one embodiment, the invention provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-PD-L1antibody, or antigen-binding portion thereof. Preferably, the antibodyis a human anti-PD-L1 antibody (such as any of the human anti-humanPD-L1 antibodies described herein). Additionally or alternatively, theantibody may be a chimeric or humanized anti-PD-L1 antibody.

Preferred cancers whose growth may be inhibited using the antibodies ofthe invention include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment includemelanoma (e.g., metastatic malignant melanoma), renal cancer, prostatecancer, breast cancer, colon cancer and lung cancer. Examples of othercancers that may be treated using the methods of the invention includebone cancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular malignant melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer,testicular cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small Intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present invention is also useful fortreatment of metastatic cancers, especially metastatic cancers thatexpress PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144).

Optionally, antibodies to PD-L1 can be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 713:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactivation by PD-L1 blockade, we may expect to activate tumor responsesin the host.

PD-L1 blockade is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayai, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, CancerPrinciples and Practice of Oncology. Fifth Edition). In one of thesestrategies, a vaccine is prepared using autologous or allogeneic tumorcells. These cellular vaccines have been shown to be most effective whenthe tumor cells are transduced to express GM-CSF. GM-CSF has been shownto be a potent activator of antigen presentation for tumor vaccination(Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90:3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so-called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. PD-L1 blockade may be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al. (1994)Science 266: 2011-2013). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (i.e. bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-L1blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot, R &Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997)Science 228:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with PD-L1 blockade to activate more potentanti-tumor responses.

PD-L1 blockade may also be combined with standard cancer treatments.PD-L1 blockade may be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is ananti-PD-L1 antibody in combination with decarbazine for the treatment ofmelanoma. Another example of such a combination is an anti-PD-L1antibody in combination with interleukin-2 (IL-2) for the treatment ofmelanoma. The scientific rationale behind the combined use of PD-L1blockade and chemotherapy is that cell death, that is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withPD-L1 blockade through cell death are radiation, surgery, and hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors may also be combined with PD-L1blockade. Inhibition of angiogenesis leads to tumor cell death which mayfeed tumor antigen into host antigen presentation pathways.

PD-L1 blocking antibodies can also be used in combination withbispecific antibodies that target Fc alpha or Fc γ receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would by augmentedby the use of PD-L1 blockade. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities may be used incombination with anti-PD-L1 to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with anti-PD-L1. These includemolecules on the surface of dendritic cells which activate DC functionand antigen presentation. Anti-CD40 antibodies are able to substituteeffectively for T cell helper activity (Ridge, J. et al. (1998) Nature393: 474-478) and can be used in conjunction with PD-L1 antibodies (Ito,N. et al. (2000) Immunobiology 201 (5) 527-40). Activating antibodies toT cell costimulatory molecules such as OX-40 (Weinberg, A. et al. (2000)Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine3: 682-685 (1997), and ICOS (Hutloff, A. et al. (1999) Nature 397:262-266) as well as antibodies which block the activity of negativecostimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097)or BTLA (Watanabe, N. et al. (2003) Nat Immunol 4:670-9), B7-H4 (Sica, GL et al. (2003) Immunity 18:849-61) may also provide for increasedlevels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. PD-L1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofanti-PD-L1 antibodies may be expected to increase the frequency andactivity of the adoptively transferred T cells.

Infectious Diseases Other methods of the invention are used to treatpatients that have been exposed to particular toxins or pathogens.Accordingly, another aspect of the invention provides a method oftreating an infectious disease in a subject comprising administering tothe subject an anti-PD-L1 antibody, or antigen-binding portion thereofsuch that the subject is treated for the infectious disease. Preferably,the antibody is a human anti-human PD-L1 antibody (such as any of thehuman anti-PD-L1 antibodies described herein). Additionally oralternatively, the antibody can be a chimeric or humanized antibody.

Similar to its application to tumors as discussed above, antibodymediated PD-L1 blockade can be used alone, or as an adjuvant, incombination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, & C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, PseudomonasAeruginosa. PD-L1 blockade is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of the infections. These novel epitopes are recognized as foreignat the time of anti-human PD-L1 administration, thus provoking a strongT cell response that Is not dampened by negative signals through PD-L1.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme's disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (Mucor, Absidia, rhizophus), Sporothrixschenkii, Blasatomyes dermatitidis, Paracocidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, Nippostrongylus brasiliensis.

In all of the above methods, PD-L1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad Sc. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Autoimmune Reactions

Anti-PD-L1 antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (depigmentation observed in anti-CTLA-4+GM-CSF-modified B16melanoma in van Elsas et al. supra; depigmentation in Trp-2 vaccinatedmice (Overwijk, W. et al. (1999) Proc. Natl. Acad Sci. U.S.A. 96:2982-2987); autoimmune prostatitis evoked by TRAMP tumor cell vaccines(Hurwitz, A. (2000) supra), melanoma peptide antigen vaccination andvitilago observed in human clinical trials (Rosenberg, S A and White, DE (1996) J. Immunother Emphasis Tumor Immunol 19(1): 81-4).

Therefore, it is possible to consider using anti-PD-L1 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of Aβ peptide in amyloid deposits inthe brain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFα for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-PD-L1 antibody. Neutralizing antibody responses toreproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-PD-L1 antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα, and IgE.

Vaccines

Anti-PD-L1 antibodies may be used to stimulate antigen-specific immuneresponses by coadministration of an anti-PD-L1 antibody with an antigenof interest (e.g., a vaccine). Accordingly, in another aspect theinvention provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject (i) theantigen; and (ii) an anti-PD-L1 antibody, or antigen-binding portionthereof, such that an Immune response to the antigen in the subject isenhanced. Preferably, the antibody is a human anti-human PD-L1 antibody(such as any of the human anti-PD-L1 antibodies described herein).Additionally or alternatively, the antibody can be a chimeric orhumanized antibody. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove, or antigens from the viruses, bacteria or other pathogensdescribed above.

Anti-PD-L1 antibodies may also be used to abrogate secondary effectsassociated with diseases such as T cell suppressed wasting disease withcolitis (Kanai et a. (2003) J. Immunol. 171:4156-63). Accordingly, inanother aspect the invention provides a method of abrogating leukocyteinfiltration, decreasing production of IFN-γ, IL-2, and IFN-α by Tcells. Preferably, the antibody is a human anti-human PD-L1 antibody(such as any of the human anti-PD-L1 antibodies described herein).Additionally or alternatively, the antibody can be a chimeric orhumanized antibody.

Anti-PD-L1 antibodies may also be used to treat diseases such as chronicinflammatory diseases, such as lichen planus, a T-cell mediated chronicinflammatory mucocutaneous disease (Youtgnak-Piboonratanakit et al.(2004) Immnol Letters 94:215-22). Accordingly, in another aspect theinvention provides a method of abrogating chronic inflammatory diseaseby T cells. Preferably, the antibody is a human anti-human PD-L1antibody (such as any of the human anti-PD-L1 antibodies describedherein). Additionally or alternatively, the antibody can be a chimericor humanized antibody.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of the invention in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-PD-L1 antibodies of the inventioncan be co-administered with one or other more therapeutic agents, e g, acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/dose once every fourweeks and adriamycin is intravenously administered as a 60-75 mg/ml doseonce every 21 days. Co-administration of the human anti-PD-L1antibodies, or antigen binding fragments thereof, of the presentinvention with chemotherapeutic agents provides two anti-cancer agentswhich operate via different mechanisms which yield a cytotoxic effect tohuman tumor cells. Such co-administration can solve problems due todevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells which would render them unreactive with the antibody.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain a least one additionalreagent, or one or more additional human antibodies of the invention(e.g., a human antibody having a complementary activity which binds toan epitope in PD-L1 antigen distinct from the first human antibody).Kits typically include a label indicating the intended use of thecontents of the kit. The term label includes any writing, or recordedmaterial supplied on or with the kit, or which otherwise accompanies thekit.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Monoclonal Antibodies AgainstPD-L1

Antigen

Immunization protocols utilized as antigen both (i) a recombinant fusionprotein comprising the extracellular portion of PD-L1, and (ii) membranebound full-length PD-L1. Both antigens were generated by recombinanttransfection methods in a CHO cell line.

Transgenic Mice (KM-Mouse® Colony

Fully human monoclonal antibodies to PD-L1 were prepared using the KMstrain of transgenic transchromosomic mice, which expresses humanantibody genes. In this mouse strain, the endogenous mouse kappa lightchain gene has been homozygously disrupted as described in Chan et al.(1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene hasbeen homozygously disrupted as described in Example 1 of PCT PublicationWO 01/09187. Furthermore, this mouse strain carries a human kappa lightchain transgene, KCo5, as described in Fishwild et al (1996) NatureBiotechnology 14:845-851, and a SC20 transchromosome as described in PCTPublication WO 02/43478.

KM-Mouse® Immunizations

To generate fully human monoclonal antibodies to PD-L1, a cohort of miceof the KM-Mouse® strain were immunized with purified recombinantPD-L1-Ig and PD-L1-transfected CHO cells as antigen. Generalimmunization schemes for HuMab mice are described in Lonberg, N. et al.(1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice were6-16 weeks of age upon the first infusion of antigen. A purifiedrecombinant preparation (5-50 μg) of PD-L1-Ig antigen and 5-10×10⁶ cellswere used to immunize the HuMab mice intraperitonealy (IP),subcutaneously (Sc) or via footpad injection.

Transgenic mice were immunized twice with antigen in complete Freund'sadjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of11 immunizations) with the antigen in Incomplete Freund's or Ribiadjuvant. The immune response was monitored by retroorbital bleeds. Theplasma was screened by ELISA (as described below), and mice withsufficient titers of anti-PD-L1 human immunogolobulin were used forfusions. Mice were boosted intravenously with antigen 3 days beforesacrifice and removal of the spleen. Typically, 10-35 fusions for eachantigen were performed. Several dozen mice were immunized for eachantigen.

Selection of KM-Mouse® Producing Anti-PD-L1 Antibodies: To select HuMabmice producing antibodies that bound PD-L1, sera from immunized micewere tested by ELISA as described by Fishwild, D. et al. (1996).Briefly, microtiter plates were coated with purified recombinant PD-L1fusion protein from transfected CHO cells at 1-2 μg/ml in PBS, 100μl/wells incubated 4° C. overnight then blocked with 200 μl/well of 5%fetal bovine serum in PBS/Tween (0.05%). Dilutions of sera fromPD-L1-immunized mice were added to each well and incubated for 1-2 hoursat ambient temperature. The plates were washed with PBS/Tween and thenincubated with a goat-anti-human IgG polyclonal antibody conjugated withhorseradish peroxidase (HRP) for 1 hour at room temperature. Afterwashing, the plates were developed with ABTS substrate (Sigma, A-1888,0.22 mg/ml) and analyzed by spectrophotometer at OD 415-495. Mice thatdeveloped the highest titers of anti-PD-L1 antibodies were used forfusions. Fusions were performed as described below and hybridomasupernatants were tested for anti-PD-L1 activity by ELISA.Generation of Hybridomas Producing Human Monoclonal Antibodies to PD-L1:The mouse splenocytes, isolated from a KM mouse, were fused with PEG toa mouse myeloma cell line based upon standard protocols. The resultinghybridomas were then screened for the production of antigen-specificantibodies. Single cell suspensions of splenocytes from immunized micewere fused to one-fourth the number of SP2/0 nonsecreting mouse myelomacells (ATCC, CRL 1581) with 50% PEG (Sigma). Cells were plated atapproximately 1×10⁵/well in flat bottom microtiter plate, followed byabout two week incubation in selective medium containing 10% fetalbovine serum, 10% P388DI (ATCC, CRL TIB-63) conditioned medium, 3-5%origen (IGEN) in DMEM (Mediatech, CRL 10013, with high glucose,L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 mg/ml gentamycin and 1×HAT (Sigma, CRL P-7185).After 1-2 weeks, cells were cultured in medium in which the HAT wasreplaced with HT. Individual wells were then screened by ELISA(described above) for human anti-PD-L1 monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. The antibody-secreting hybridomas were re-plated, screenedagain and, if still positive for human IgG, anti-PD-L1 monoclonalantibodies were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for further characterization.

Hybridoma clones 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7,and 13G4 were selected for further analysis.

Example 2 Structural Characterization of Human Monoclonal Antibodies3G10, 12A4, and 10A5

The cDNA sequences encoding the heavy and light chain variable regionsof the 3G10, 12A4, 10A5, 5F8, 10H1, 1B12, 7H1, 11E6, 12B7, and 13G4monoclonal antibodies were obtained from the 3G10, 12A4, 10A5, 5F8,10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 hybridomas, respectively, usingstandard PCR techniques and were sequenced using standard DNA sequencingtechniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 3G10 are shown in FIG. 1A and in SEQ ID NO:81 and 1,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 3G10 are shown in FIG. 1B and in SEQ ID NO:91 and 11,respectively.

Comparison of the 3G10 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 3G10 heavy chain utilizes a VH segment from human germline VH 1-18,an undetermined D segment, and a JH segment from human germline JH 6b.The alignment of the 3G10 VH sequence to the germline VH 1-18 sequenceis shown in FIG. 11. Further analysis of the 3G10 VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 1A and 11, andin SEQ ID NO:21, 31 and 41, respectively.

Comparison of the 3G10 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 3G10 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 1. The alignment of the 3G10 VLsequence to the germline VK L6 sequence is shown in FIG. 21. Furtheranalysis of the 3G010 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 1B and 21, and in SEQ ID NOs:51, 61 and71, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 12A4 are shown in FIG. 2A and in SEQ ID NO:82 and 2,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 12A4 are shown in FIG. 2B and in SEQ ID NO:92 and 12,respectively.

Comparison of the 12A4 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 12A4 heavy chain utilizes a VH segment from human germline VH 1-69,a D segment from human germline 3-10, and a JH segment from humangermline JH 6b. The alignment of the 12A4 VH sequence to the germline VH1-69 sequence is shown in FIG. 12. Further analysis of the 12A4 VHsequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 2A and 12, and in SEQ ID NOs:22, 32 and 42, respectively.

Comparison of the 12A4 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 12A4 light chain utilizes a VL segment from human germline VK 16 anda JK segment from human germline JK 1. The alignment of the 12A4 VLsequence to the germline VK L6 sequence is shown in FIG. 22. Furtheranalysis of the 12A4 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 2B and 22, and in SEQ ID NOs:52, 62 and72, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 10A5 are shown in FIG. 3A and in SEQ ID NO:83 and 3,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 10A5 are shown in FIG. 3B and in SEQ ID NO:93 and 13,respectively.

Comparison of the 10A5 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 10A5 heavy chain utilizes a VH segment from human germline VH 1-3, aD segment from human germline 5-5, and a JH segment from human germlineJH 4b. The alignment of the 10A5 VH sequence to the germline VH 1-3sequence is shown in FIG. 13. Further analysis of the 10A5 VH sequenceusing the Kabet system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 3A and 13, and in SEQ ID NOs:23, 33, and 43, respectively.

Comparison of the 10A5 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 10A5 light chain utilizes a VL segment from human germline VK L15and a JK segment from human germline JK 2. The alignment of the 10A5 VLsequence to the germline VK L15 sequence is shown in FIG. 23. Furtheranalysis of the 10A5 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3B and 23, and in SEQ ID NOs:53, 63, and73, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 5F8 are shown in FIG. 4A and in SEQ ID NO:84 and 4,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 5F8 are shown in FIG. 4B and in SEQ ID NO:94 and 14,respectively.

Comparison of the 5F8 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 5F8 heavy chain utilizes a VH segment from human germline VH 1-69, aD segment from human germline 6-13, and a JH segment from human germlineJH 4b. The alignment of the 5F8 VH sequence to the germline VH 1-69sequence is shown in FIG. 14. Further analysis of the 5F8 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 4A and 14, and in SEQ ID NOs:24, 34, and 44, respectively.

Comparison of the 5F8 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 5F8 light chain utilizes a VL segment from human germline VK A27 anda JK segment from human germline JK 1. The alignment of the 5F8 VLsequence to the germline VK A27 sequence is shown in FIG. 24. Furtheranalysis of the 5F8 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 4B and 24, and in SEQ ID NOs:54, 64, and74, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 10H10 are shown in FIG. 5A and in SEQ ID NO:85 and 5,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 10H10 are shown in FIG. 5B and in SEQ ID NO:95 and 15,respectively.

Comparison of the 10H10 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 10H10 heavy chain utilizes a VH segment from human germline VH 3-9,a D segment from human germline 4-17, and a JH segment from humangermline JH 4b. The alignment of the 10H10 VH sequence to the germlineVH 3-9 sequence is shown in FIG. 15. Further analysis of the 10H10 VHsequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFigures SA and 15, and in SEQ ID) NOs:25, 35, and 45, respectively.

Comparison of the 10H10 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 10H10 light chain utilizes a VL segment from human germline VK L15and a JK segment from human germline JK 2. The alignment of the 10H10 VLsequence to the germline VK L15 sequence is shown in FIG. 25. Furtheranalysis of the 10H10 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in Figures SB and 25, and in SEQ ID NOs:55, 65, and75, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 1B12 are shown in FIG. 6A and in SEQ ID NO:86 and 6,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 1B12 are shown in FIG. 6B and in SEQ ID) NO:96 and 16,respectively.

Comparison of the 1B12 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 1B12 heavy chain utilizes a VH segment from human germline VH 1-69,a D segment from human germline 3-10, and a JH segment from humangermline JH 6b. The alignment of the 1B12 VH sequence to the germline VH1-69 sequence is shown in FIG. 16. Further analysis of the 1B12 VHsequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 6A and 16, and in SEQ ID NOs:26, 36, and 46, respectively.

Comparison of the 1B12 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 1B12 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 1. The alignment of the 1B12 VLsequence to the germline VK L6 sequence is shown in FIG. 26. Furtheranalysis of the 1B12 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 61 and 26, and in SEQ ID NOs:56, 66, and76, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 7H1 are shown in FIG. 7A and in SEQ ID NO:87 and 7,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 7H1 are shown in FIG. 7B and in SEQ ID NO:97 and 17,respectively.

Comparison of the 7H1 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 7H1 heavy chain utilizes a VH segment from human germline VH 1-69, aD segment from human germline 3-10, and a JH segment from human germlineJH 6b. The alignment of the 7H1 VH sequence to the germline VII 1-69sequence is shown in FIG. 17. Further analysis of the 7H1 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 7A and 17, and in SEQ ID NOs:27, 37, and 47, respectively.

Comparison of the 7H1 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 7H1 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 1. The alignment of the 7H1 VLsequence to the germline VK L6 sequence is shown in FIG. 27. Furtheranalysis of the 7H1 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 7B and 27, and in SEQ ID NOs:57, 67, and77, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 11E6 are shown in FIG. 4A and in SEQ ID NO:84 and 4,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 11E6 are shown in FIG. 41 and in SEQ ID NO:94 and 14,respectively.

Comparison of the 11E6 heavy chain Immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 11E6 heavy chain utilizes a VH segment from human germline VH 1-69,a D segment from human germline 6-19, and a JH segment from humangermline JH 6c. The alignment of the 11E6 VH sequence to the germline VH1-69 sequence is shown in FIG. 18. Further analysis of the 11E6 VHsequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 8A and 18, and in SEQ ID NOs:28, 38, and 4, respectively.

Comparison of the 11E6 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 11E6 light chain utilizes a VL segment from human germline VK A27and a JK segment from human germline JK 4. The alignment of the 11E6 VLsequence to the germline VK A27 sequence is shown in FIG. 27. Furtheranalysis of the 11E6 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 8B and 28, and in SEQ ID NOs:58, 68, and78, respectively. In addition, a second related clone included the VKsequence as shown in SEQ ii) NO:109. This antibody is denoted herein as11E6a.

The nucleotide and amino acid sequences of the heavy chain variableregion of 12B7 are shown in FIG. 9A and in SEQ ID NO:89 and 9,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 12B7 are shown in FIG. 9B and in SEQ ID NO:99 and 19,respectively.

Comparison of the 12B7 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 12B7 heavy chain utilizes a VH segment from human germline VH 1-69,a D segment from human germline 3-10, and a JH segment from humangermline JH 6b. The alignment of the 12B7 VH sequence to the germline VH1-69 sequence is shown in FIG. 19. Further analysis of the 12B7 VHsequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 9A and 19, and in SEQ ID NOs:29, 39, and 49, respectively.

Comparison of the 12B7 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 12B7 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 5. The alignment of the 12B7 VLsequence to the germline VK L6 sequence is shown in FIG. 29. Furtheranalysis of the 12B7 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 9B and 29, and in SEQ ID NOs:59, 69, and79, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 13G4 are shown in FIG. 10A and in SEQ ID NO:90 and 10,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 13G4 are shown in FIG. 10B and in SEQ ID NO:100 and 20,respectively.

Comparison of the 13G4 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 13G4 heavy chain utilizes a VH segment from human germline VH 3-9, aD segment from human germline 3-9, and a JH segment from human germlineJH 4b. The alignment of the 13G4 VH sequence to the germline VH 3-9sequence is shown in FIG. 20. Further analysis of the 13G4 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 10A and 20, and in SEQ ID NOs:30, 40, and 50, respectively.

Comparison of the 13G4 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 13G4 light chain utilizes a VL segment from human germline VK L18and a JK segment from human germline JK 3. The alignment of the 13G4 VLsequence to the germline VK L18 sequence is shown in FIG. 30. Furtheranalysis of the 13G4 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 10B and 30, and in SEQ ID NOs:60, 70, and80, respectively.

Example 3 Characterization of Binding Specificity and Binding Kineticsof Anti-PD-L1 Human Monoclonal Antibodies

In this example, binding affinity and binding kinetics of anti-PD-L1antibodies were examined by Biacore analysis. Binding specificity, andcross-competition were examined by flow cytometry.

Binding Affinity and Kinetics

Anti-PD-L1 antibodies were characterized for affinities and bindingkinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Purifiedrecombinant human PD-L1 fusion protein was covalently linked to a CM5chip (carboxy methyl dextran coated chip) via primary amines, usingstandard amine coupling chemistry and kit provided by Biacore, to adensity of 562 RUs. Binding was measured by flowing the antibodies inHBS EP buffer (provided by Biacore AB) at a concentration of 133 nM at aflow rate of 50 μl/min. The antigen-antibody association kinetics wasfollowed for 1 minute and the dissociation kinetics was followed for 1minute. The association and dissociation curves were fit to a 1:1Langmuir binding model using BIAevaluation software (Biacore AB). Tominimize the effects of avidity in the estimation of the bindingconstants, only the initial segment of data corresponding to associationand dissociation phases were used for fitting. The K_(d), k_(on) andk_(off) values that were determined are shown in Table 2.

TABLE 2 Biacore binding data for PD-L1 human monoclonal antibodiesAffinity K_(D) × On rate k_(on) × Off rate k_(off) × Sample # Sample ID10⁻⁹ (M) 10⁵ (1/Ms) 10⁻⁴ 1/s 1 3G10 3.39 5.25 17.8 3 10A5 1.45 2.58 3.72Additional binding data obtained by equilibrium binding method andanalyzed on GraphPad Prizm is shown in Table 3.

TABLE 3 Biacore equilibrium binding data for PD-L1 human monoclonalantibodies. K_(D) K_(D) Clone (nM) (nM) ID 37 C. 25 C. 12A4 1.94 0.767H1 2.15 nd 1B12 1.38 0.61 12B7 0.83 0.53 10A5 2.41 0.57 10H10 5.93 5.4813G4 1.87 3.3 11E6 0.53 2.9 5F8 2.17 0.75Binding Specificity by Flow Cytometry

Chinese hamster ovary (CHO) cell lines that express recombinant humanPD-L1 at the cell surface were developed and used to determine thespecificity of PD-L1 human monoclonal antibodies by flow cytometry. CHOcells were transfected with expression plasmids containing full lengthcDNA encoding transmembrane forms of PD-L1. Binding of the 3G10, 10A5,and 12A4 anti-PD-L1 human monoclonal antibodies was assessed byincubating the transfected cells with the anti-PD-L1 human monoclonalantibody. The cells were washed and binding was detected with aFITC-labeled anti-human IgG Ab. Flow cytometric analyses were performedusing a FACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Thebinding was compared to the parent CHO cell line. The results are shownin FIGS. 32A (HuMAb 3G10), 32B (HuMAb 10A5) and 32C (HuMAb 12A4).Binding was also tested using varying concentrations of an anti-PD-L1antibody. The results are shown in FIG. 33. The anti-PD-L1 humanmonoclonal antibodies 3G10, 10A5, and 12A4 bound to the CHO cellstransfected with PD-L1 in a concentration dependent manner. These datademonstrate that the anti-PD-L1 human monoclonal antibodies specificallybind to cell surface PD-L1.

Binding Specificity by ELISA

The specificity of the anti-PD-L1 monoclonal antibodies was determinedusing a standard ELISA assay for binding to a human PD-L1 fusion to animmunoglobulin Fe region.

An Fc-fusion protein of human PD-L1 was tested for binding against theanti-PD-L1 human monoclonal antibodies 3G10, 12A4, and 10A5. StandardELISA procedures were performed. The anti-PD-L1 human monoclonalantibodies were added at different concentrations. Goat-anti-human IgG(kappa chain-specific) polyclonal antibody conjugated with horseradishperoxidase (HRP) was used as secondary antibody. The results are shownin FIG. 34. Each of the anti-PD-L11 human monoclonal antibodies 3G10,12A4, and 10A5 bound with high specificity to PD-L1.

Example 4 Characterization of Anti-PD-L1 Antibody Binding to PD-L1Expressed on the Cell Surface of Human and Monkey T Cells

Anti-PD-L1 antibodies were tested by flow cytometry for binding toactivated human or cynomolgus monkey T cells expressing PD-L1 on theirsurface.

Human or monkey T cells were activated by anti-CD3 antibody to inducePD-L1 expression prior to binding with a human anti-PD-L1 monoclonalantibody. Binding of the 3G10, 1B12, 13G4, and 12A4 anti-PD-L1 humanmonoclonal antibodies was assessed by incubating the activated cellswith serial dilutions of the anti-PD-L1 human monoclonal antibodies. Anisotype control antibody was used as a negative control. The cells werewashed and binding was detected with a FITC-labeled anti-human Ig-kappalight chain Ab. Flow cytometric analyses were performed using aFACScalibur flow cytometer (Becton Dickinson, San Jose, Calif.). Theresults are shown in FIGS. 35 and 36. The anti-PD-L1 monoclonalantibodies 3G10, 1B12, 13G4, and 12A4 bound to activated human andmonkey T cells. These data demonstrate that the anti-PD-L1 humanmonoclonal antibodies bind to human and cynomolgus monkey cell surfacePD-L1.

Example 5 Characterization of Anti-PD-L1 Antibody Binding to PD-L1Expressed on the Cell Surface of Human T Cells

Anti-PD-L1 antibodies were tested for binding to activated human T cellsexpressing PD-L1 on their cell surface by flow cytometry.

Human T cells were activated by anti-CD3 antibody to induce PD-1,expression on T cells prior to binding with a human anti-PD-L1monoclonal antibody. Binding of the 3G10, 10A5 and 12A4 anti-PD-L1 humanmonoclonal antibodies was assessed by incubating the activated T cellswith the anti-PD-L1 human monoclonal antibodies at a concentration of 20μg/ml. An isotype control antibody was used as a negative control. Thecells were washed and binding was detected with a FITC-labeledanti-human IgG Ab. Flow cytometric analyses were performed using aFACScalibur flow cytometry (Becton Dickinson, San Jose, Calif.). Theresults are shown in FIGS. 37A (HuMAb 3G10), 37B (HuMAb 10A5) and 37C(HuMAb 12A4). The anti-PD-L1 monoclonal antibodies 3G10, 10A5, and 12A4bound to activated human T cells (bold line), as shown in histogramplots compared to control (light line). These data demonstrate that theanti-PD-L1 human monoclonal antibodies bind to human cell surface PD-L1.

Example 6 Binding Specificity by Flow Cytometry

The ES-2 human ovarian carcinoma cell line that expresses human PD-L1 atthe cell surface was used to determine the specificity of PD-L1 humanmonoclonal antibodies by flow cytometry. ES-2 cells were treatedovernight with 500 IU/mL of recombinant hIFN-γ to increase PD-L1expression over the basal level. Binding of the 12A4, 1B12, 3G10, 10A5,12B7, 13G4, 11B6, and 5F8 anti-PD-L1 human monoclonal antibodies wasassessed by incubating the induced cells with serial dilutions of theanti-PD-L1 human monoclonal antibody. The cells were washed and bindingwas detected with a PB-labeled anti-human IgG Ab. Flow cytometricanalyses were performed using a FACScalibur flow cytometer (BectonDickinson, San Jose, Calif.). The binding was compared to isotypecontrol antibody. The results are shown in FIG. 38. The anti-PD-L1 humanmonoclonal antibodies 12A4, 1B12, 3G10, 10A5, 12B7, 13G4, 11E6, and 5F8bound to the hIFN-γ-induced ES-2 cells in a concentration dependentmanner. These data demonstrate that the anti-PD-L1 human monoclonalantibodies specifically bind to cell surface PD-L1.

Example 7 Effect of Human Anti-PD-L1 Antibodies on Cell Proliferationand Cytokine Production in a Mixed Lymphocyte Reaction

A mixed lymphocyte reaction was employed to demonstrate the effect ofblocking the PD-L1/PD-1 pathway to lymphocyte effector cells. T cells inthe assay were tested for proliferation, IFN-γ secretion and IL-2secretion in the presence or absence of an anti-PD-L1 human monoclonalantibody.

Human CD4+T-cells were purified from PBMC using a CD4+ positiveselection kit (Dynal Biotech). Dendritic cells were derived frompurified monocytes cultured with 1000 U/ml of L-4 and 500 U/ml of GM-CSF(R&D Biosystems) for seven days. Monocytes were prepared using amonocyte negative selection kig (Mitenyi Biotech). Each culturecontained 10⁵ purified T-cells and 10⁶ allogeneic dendritic cells in atotal volume of 200 μl. Anti-PD-L1 monoclonal antibody 10A5, 12A4, or3G10 was added to each culture at different antibody concentrations.Either no antibody or an isotype control antibody was used as a negativecontrol. The cells were cultured for 5 days at 37° C. After day 5, 100μl of medium was taken from each culture for cytokine measurement. Thelevels of IFN-γ and IL-2 were measured using OptEIA ELISA kits (BDBiosciences). The cells were labeled with ³H-thymidine, cultured foranother 18 hours, and analyzed for cell proliferation. The results areshown in FIGS. 39A (T cell proliferation), 39B (IFN-γ secretion usingHuMAb 10A5), 39C (IFN-γ secretion using HuMAb 12A4 or 3G10) and 39D(IL-2 secretion). The anti-PD-L1 human monoclonal antibody 10A5 promotesT-cell proliferation, IFN-γ secretion and IL-2 secretion in aconcentration dependent manner. The anti-PD-L1 human monoclonalantibodies 12A4 and 3G10 also showed an increase in IFN-γ secretion. Incontrast, cultures containing the control antibody did not show anincrease in T cell proliferation, IFN-γ or IL-2 secretion.

In a separate experiment, an allogeneic mixed lymphocyte reaction (MLR)was employed to demonstrate the effect of blocking the PD-L1/PD-1pathway in lymphocyte effector cells. T cells in the assay were testedfor proliferation and IFN-γ secretion in the presence or absence of ananti-PD-L1 human monoclonal antibody or isotype control antibody.

Human CD4+T-cells were purified from PBMC using a CD4+ negativeselection kit (Miltenyi). Monocytes were prepared using a monocytenegative selection kit (Mitenyi Biotech). Dendritic cells were derivedfrom purified monocytes cultured with 1000 U/ml of IL-4 and 500 U/ml ofGM-CSF (R&D Biosystems) for seven days. Each MLR culture contained 10⁵purified T-ells and 10⁴ allogeneic dendritic cells in a total volume of200 μl. Anti-PD-L1.1 monoclonal antibody 12A4, 11E6, 3G10, 13G4, 1B12,10A5, and 12B7 were added to each culture at different antibodyconcentrations. Either no antibody or an isotype control antibody wasused as a negative control. The cells were cultured for 5 days at 37° C.On day 5, 50 μl of medium was taken from each culture for cytokinemeasurement and replaced with an equal volume of culture mediumcontaining 1 μCi of ³H-thymidine. The cells were cultured for another 18hours, harvested, and analyzed for cell proliferation. The levels ofIFN-γ in the culture fluid were measured using an OptEIA hIFN-γ ELISAkit (BD Biosciences). The results are shown in FIG. 40. The anti-PD-L1human monoclonal antibodies promote T-cell proliferation and IFN-γsecretion in a concentration-dependent manner. In contrast, culturescontaining the control antibody did not show an increase in T cellproliferation or IFN-γ secretion.

Example 8 Effect of Human Anti-PD-L1 Antibody on Function of TRegulatory Cells

T regulatory cells (CD4+, CD25+) are lymphocytes that suppress theimmune response. The effect of the addition of T regulatory cells onproliferation and IFN-γ secretion in the allogeneic dendritic cell and Tcell MLR in the presence or absence of an anti-PD-L1 human monoclonalantibody was tested.

T regulatory cells were purified from PBMC using a CD44CD25+ regulatoryT cell isolation kit (Miltenyi Biotec). T regulatory cells were addedinto a mixed lymphocyte reaction (see above) containing purifiedCD4+CD25-T cells and allogeneic dendritic cells in a 2:1 ratio ofCD4+CD25- to T regulatory cells. Anti-PD-L1 monoclonal antibody 10A5 wasadded to each culture at a concentration of 10 μg/ml. Either no antibodyor an isotype control antibody was used as a negative control. The cellswere cultured for 5 days at 37′C at which time the supernatants wereanalyzed for IFN-γ secretion using a Beadlyte cytokine detection system(Upstate). The cells were labeled with ³H-thymidine, cultured foranother 18 hours, and analyzed for cell proliferation. The results areshown in FIGS. 41A (T cell proliferation) and 41B (IFN-γ secretion). Theaddition of anti-PD-L1 human monoclonal antibody 10A5 promotes both Tcell proliferation and IFN-γ secretion in cell cultures of allogeneicdendritic cells, T cells and T regulatory cells, indicating thatanti-PD-L1 antibodies can reverse the effect of T regulatory cells inthe allogeneic DC-T cell-MLR

In a separate experiment, human anti-PD-L1 antibodies 12A4 and 13G4, anda control antibody 1D12, were tested in the MLR assay with T regulatorycells. The results are shown in FIGS. 42 (T cell proliferation) and 43(IFN-γ secretion). The addition of anti-PD-L) human monoclonalantibodies 12A4 or 13G4 partially reverses the suppression of both Tcell proliferation and IFN-γ secretion in cell cultures of allogeneicdendritic cells and T cells containing T-regulatory cells, indicatingthat anti-PD-L1 antibodies may have an effect on T-regulatory cells.

Example 9 Effect of Anti-PD-1 Antibodies on Cytokine Secretion by ViralAntigen-Stimulated PBMC Cells from a Positive CMV Responsive Donor

CMV antigen-responsive human PBMC (Astarte Biologics, Redmond, Wash.)were cultured at 2e5 cells/well in flat bottom TC-treated 96 wellplates, in the presence of 0.5 ug/ml CMV lysate (Astarte Biologics)+/−titrated anti-PD-L1 antibodies. AIM-V medium (Invitrogen) supplementedwith heat-inactivated FBS (10% final) was used at a total volume of 200ul/well. The cells were cultured for 4 days at 37° C., 5% CO₂ at whichtime culture supernatant was harvested for determination of secretedinterferon-γ by ELISA (OptEIA hIFN-γ ELISA kit-BD Biosciences). Theresults are shown in FIG. 44. The anti-PD-L1 human monoclonal antibodiespromote IFN-γ secretion by CMV-specific T-cells in a dose-dependentmanner. The most robust response was generated by antibodies 13G4, 1B12,and 12A4 compared to isotype control. These results shows thatanti-PD-L1 HuMAbs can stimulate IFN-γ release in a memory T cellresponse from PBMC cells previously stimulated against an antigen.

Example 10 Blocking of PD-L1 Ligand Binding to PD-1 by Human Anti-PD-L1Antibodies

Anti-PD-L1 human monoclonal antibodies were tested for the ability toblock binding of the ligand PD-L1 to PD-1 expressed on transfected CHOcells by using a cell cytometry assay.

PD-1 expressing CHO cells were suspended in FACS buffer (PBS with 4%fetal calf serum). Various concentrations of the anti-PD-L1 HuMAbs 3G10,10A5 or 12A4 was added to the cell suspension tubes at 4° C. for 30minutes, followed by addition FITC-labeled PD-L1 fused to animmunoglobulin Fc-region. Flow cytometric analyses were performed usinga FACScalibur flow cytometer (Becton Dickinson, San Jose, Calif.). Theresults are depicted in FIG. 45. The anti-PD-L1 monoclonal antibodies3G10, 10A5, and 12A4 blocked binding of PD-L1 to CHO cells transfectedwith human PD-1, as measured by the mean fluorescent intensity (MFI) ofstaining. These data demonstrate that the anti-PD-L1 HuMAbs blockbinding of PD-L1 ligand to cell surface PD-1.

Example 11 Inhibition of the Binding of Soluble PD-1 to Cell-SurfacePD-L1 by Human Anti-PD-L1 Antibodies

Anti-PD-L1 human monoclonal antibodies were tested for the ability toblock binding of a soluble dimeric version of the PD-1 receptor(PD-1-hFc) to PD-L1 expressed on hIFN-γ-induced BS-2 human ovariancarcinoma cells using a flow cytometry assay. The blocking was comparedto isotype control antibody.

ES-2 cells were induced overnight with 500 IU/mL of hIFN-γ to upregulatehPD-L1 cell surface expression. Induced cells were suspended in FACSbuffer. Serial dilutions of the anti-PD-L1 HuMAbs 12A4, 1B12, 3G10,10A5, 12B7, 13G4, 11E6, and 5F8 were added to the cell suspension tubesat 4° C. for 30 minutes, followed by two washes to remove unboundantibody. Next PD-1-hFc protein was added at a constant 2 ug/mL to allwells at 4° C. for 30 minutes, followed by two washes to remove unboundPD-1-hFc. Next bound PD-1-Fc was detected on the FS-2 cells by additionof biotinylated-non-blocking anti-PD-1 HuMab 26D5, which binds to PD-1when bound to PD-L1, at 4° C. for 30 minutes, followed by two washes toremove unbound antibody. Finally, bound 26D5 antibody was detected byaddition of streptavidin-PE conjugate at 4° C. for 30 minutes, followedby two washes to remove unbound conjugate. Flow cytometric analysis wasperformed using a FACScalibur flow cytometer (Becton Dickinson, SanJose, Calif.). The results are depicted in FIG. 46. The anti-PD-L1monoclonal antibodies 12A4, 1B12, 3G10, 1A5, 12B7, 13G4, 11E6, and 5F8blocked binding of PD-1 to ES-2 cells that express human PD-L1, asmeasured by the geometric mean fluorescent intensity (GMFI) of staining.These data demonstrate that the anti-PD-L1 HuMAbs block binding ofsoluble PD-1 receptor to cell surface PD-L1.

Example 12 Treatment of In Vivo Tumor Model Using Anti-PD-L1 Antibodies

Mice implanted with a cancerous tumor are treated in vivo withanti-PD-L1 antibodies to examine the in vivo effect of the antibodies ontumor growth. For the tumor studies, female AJ mice between 6-8 weeks ofage (Harlan Laboratories) are randomized by weight into 6 groups. Themice are implanted subcutaneously in the right flank with 2×10⁶ SAl/Nfibrosarcoma cells dissolved in 200 μl of DMEM media on day 0. The miceare treated with PBS vehicle, or anti-PD-L1 antibodies at 10 mg/kg. Theanimals are dosed by intraperitoneal injection with approximately 200 μlof PBS containing antibody or vehicle on days 1, 4, 8 and 11. Each groupcontains 10 animals and the groups consist of: (i) a vehicle group, (ii)control mouse IgG, and (iii) an anti-PD-L1 antibody. The mice aremonitored twice weekly for tumor growth for approximately 6 weeks. Usingan electronic caliper, the tumors are measured three dimensionally(height×width×length) and tumor volume is calculated. Mice areeuthanized when the tumors reached tumor end point (1500 mm³) or showgreater than 15% weight loss.

Example 13 In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 andAnti-PD-L1 Antibodies) on Tumor Establishment and Growth

MC38 colorectal cancer cells (available from Dr. N. Restifo, NationalCancer Institute, Bethesda, Md.; or Jeffrey Schlom, National Institutesof Health, Bethesda, Md.) were implanted in C57BL/6 mice (2×10⁶cells/mouse) and selected for treatment when tumors reached a size of100-200 mm³). On day 0 (i.e., the first day of treatment), each of fourgroups of 10 mice each was injected intraperitoneally (IP) with one ofthe following (1) 10 mg/kg mouse IgG and 10 mg/kg of rat IgG (control),(2) 10 mg/kg anti-CTLA-4 monoclonal antibody 9D9 (mouse anti-mouseCTLA-4, obtained from J. Allison, Memorial Sloan-Kettering CancerCenter, New York, N.Y.) and 10 mg/kg rat IgG, (3) anti-PD-L1 monoclonalantibody MIH5 (rat anti-mouse PD-L1, eBioscience) and 10 mg/kg mouseIgG, or (4) 10 mg/kg anti-CTLA-4 antibody 9D9 and 10 mg/kg anti-PD-L1antibody MIH5. Antibody injections were then further administered ondays 3 and, 6. Using an electronic caliper, the tumors were measuredthree dimensionally (height×width×length) and tumor volume wascalculated. Mice were euthanized when the tumors reached a designatedtumor end-point. The results are shown in FIG. 47.

This study indicates that, in the MC38 murine tumor model, anti-PD-L1antibody treatment alone has a modest effect on tumor growth resultingin a delay of tumor growth while anti-CTLA-4 has little effect in thismodel. However, the combination treatment of CTLA-4 antibody and PD-L1antibody has a significantly greater effect on tumor growth and resultsin tumor free mice.

Example 14 Immunohistochemistry Using Anti-PD-L1 Antibodies

To assess the tissue binding profiles of HuMab anti-PD-L1, unmodified12A4, 13G4, 3G10 and 12B7 were examined in a panel of normal(non-neoplastic) human tissues, Including spleen, tonsil, cerebrum,cerebellum, heart, liver, lung, kidney, pancreas, pituitary, skin, andsmall intestine, as well as lung carcinoma tissues (1 sample/each). ES-2cells were used as positive control. Hu-IgG₁ and Hu-IgG₄ were used asisotype control antibodies.

Snap frozen and OCT embedded normal and tumor tissues were purchasedfrom Cooperative Human Tissue Network (Philadelphia, Pa.) or NationalDisease Research Institute (Philadelphia, Pa.). Cryostat sections at 5μm were fixed with acetone for 10 min at room temperature, and stored at−80° C. until use. A Medarex developed immunohistochemistry protocol wasperformed using unmodified HuMab anti-PD-L1 by pre-complex of theprimary antibodies (12A4, 13G4, 3G10 and 12B7) and secondary antibody(FITC conjugated Fab fragment of goat anti-Hu-IgG. JacksonImmunoResearch Laboratories. West Grove, Pa.) before applying onto thesections. Briefly, 1 μg/ml or 5 μg/ml of the un-conjugated primaryantibodies were mixed with 3 fold excess of secondary antibodyrespectively and incubated for 30 min at room temperature, and thenexcess human gamma globulin was added for another 30 min to block theunbound secondary antibody. In parallel, isotype control antibodiesHu-IgG₁ or Hu-IgG₄ were pre-complexed in the same manner. Slides werewashed with PBS (Sigma, St. Louis, Mo.) twice, and then incubated withperoxidase block supplied in Dako EnVision+System (Dako. Carpinteria,Calif.) for 10 minutes. After two washes with PBS, slides were incubatedwith Dako protein block to block the non-specific binding sites.Subsequently, the pre-complex of primary antibodies or isotype controlswere applied onto sections and incubated for 1 hr. Following threewashes with PBS, slides were incubated with mouse anti-FITC antibody (20μg/ml. Sigma) for 30 min. After another three washes with PBS, theslides were incubated with the peroxidase-conjugated anti-mouse IgGpolymer supplied in the Dako EnVision+System for 30 min. Finally, slideswere washed as above and reacted with DAB substrate-chromogen solutionsupplied in the Dako EnVision+System for 6 min. Slides were then washedwith deionized water, counterstained with Mayer's hematoxylin (Dako),dehydrated, cleared and coverslipped with Permount (Fischer Scientific,Fair Lawn, N.J.) following routine histological procedure.

Weak to moderate staining was observed in ES-2 cells, as well as intumor cells of lung carcinoma tissues. In tonsil sections, strongstaining was seen in crypt epithelium that is heavily infiltrated bylymphoid cells, but not in the mucous stratified squamous epithelialcells. Moderate staining was seen in some cells in the inter-follicularregion, and very weak staining was seen in scattered large cells(dendritic reticulum-like cells) in the germinal center. In lung, weakstaining was found in alveoli macrophages. The staining patterns intonsil and lung tissues were similarly seen in immunohistochemistrysections using commercial anti-PD-L1 mAb (eBiosciences, San Diego,Calif.). There was overall less Intense staining by HuMabs, especiallyfor the staining in the germinal centers. In spleen, diffuse weakimmunoreactivity in red pulp was slightly above the background staining.In addition, weak to moderate staining was displayed in Kupffer-likecells in liver and scattered cells in Peyer's patch, as well as inscattered macrophage-like cells and fibroblasts mainly in focal regionof the muscularis externa of small intestine.

In cerebellum, cerebrum, heart, kidney, pancreas, pituitary and skintissues, no meaningful staining was observed when stained with all fouranti-PD-L1 HuMabs. No evident difference in staining was noted amongthose four antibodies except 12B7 and/or 3G10 displayed slightlystronger staining in liver and ES-2 cells.

PD-L1 Antibody Summary SEQ SEQ ID NO: SEQUENCE ID NO: SEQUENCE 1 VH a.a.3G10 26 VH CDR1 a.a. 1B12 2 VH a.a. 12A4 27 VH CDR1 a.a. 7H1 3 VH aa10A5 28 VH CDR1 a.a. 11E6 4 VH a.a. 5F8 29 VH CDR1 a.a. 12B7 5 VH a.a.10H10 30 VH CDR1 a.a. 13G4 6 VH a.a. 1B12 7 VH a.a. 7H1 31 VH CDR2 a.a.3G10 8 VH a.a. 11E6 32 VH CDR2 a.a. 12A4 9 VH a.a. 12B7 33 VH CDR2 a.a.10A5 10 VH a.a. 13G4 34 VH CDR2 a.a. 5F8 35 VH CDR2 a.a. 10H10 11 VKa.a. 3G10 36 VH CDR2 a.a. 1B12 12 VK a.a. 1244 37 VH CDR2 a.a. 7H1 13 VKa.a. 10A5 38 VH CDR2 a.a. 11E6 14 VK a.a. 5F8 39 VH CDR2 a.a. 12B7 15 VKa.a. 10H10 40 VH CDR2 a.a. 13G4 16 VK a.a. 1B12 17 VK a.a. 7H1 41 VHCDR3 a.a. 3G10 18 VK a.a. 11E6 42 VH CDR3 a.a. 12A4 19 VK a.a. 12B7 43VH CDR3 a.a. 10A5 20 VK a.a. 13G4 44 VH CDR3 a.a. 5F8 45 VH CDR3 a.a.10H10 21 VH CDR1 a.a. 3G10 46 VH CDR3 a.a. 1B12 22 VH CDR1 a.a. 12A4 47VH CDR3 a.a. 7H1 23 VH CDR1 a.a. 10A5 48 VH CDR3 a.a. 11E6 24 VH CDR1a.a. 5F8 49 VH CDR3 a.a. 12B7 25 VH CDR1 a.a. 10H10 50 VH CDR3 a.a. 13G451 VK CDR1 a.a. 3G10 79 VK CDR3 a.a. 12B7 52 VK CDR1 a.a. 12A4 80 VKCDR3 a.a. 13G4 53 VK CDR1 a.a. 10A5 54 VK CDR1 a.a. 5F8 81 VH n.t. 3G1055 VK CDR1 a.a. 10H10 82 VH n.t. 12A4 56 VK CDR1 a.a. 1B12 83 VH n.t.10A5 57 VK CDR1 a.a. 7H1 84 VH n.t. 5F8 58 VK CDR1 a.a. 11E6 85 VH n.t.10H10 59 VK CDR1 a.a. 12B7 86 VH n.t. 1B12 60 VK CDR1 a.a. 13G4 87 VHn.t. 7H1 88 VH n.t. 11E6 : 61 VK CDR2 a.a. 3G10 89 VH n.t. 12B7 62 VKCDR2 a.a. 12A4 90 VH n.t. 13G4 63 VK CDR2 a.a. 10A5 64 VK CDR2 a.a. 5F891 VK n.t. 3G10 65 VK CDR2 a.a. 10H10 92 VK n.t. 12A4 66 VK CDR2 a.a.1B12 93 VK n.t. 10A5 67 VK CDR2 a.a. 7H1 94 VK n.t. 5F8 68 VK CDR2 a.a.11E6 95 VK n.t. 10H10 69 VK CDR2 a.a. 12B7 96 VK n.t. 1B12 70 VK CDR2a.a. 13G4 97 VK n.t. 7H1 98 VK n.t. 11E6 71 VK CDR3 a.a. 3G10 99 VK n.t.12B7 72 VK CDR3 a.a. 12A4 100 VK n.t. 13G4 73 VK CDR3 a.a. 10A5 74 VKCDR3 a.a. 5F8 101 VH 1-18 germline a.a. 75 VK CDR3 a.a. 10H10 102 VH1-69 germline a.a. 76 VK CDR3 a.a. 1B12 103 VH 1-3 germline a.a. 77 VKCDR3 a.a. 7H1 104 VH 3-9 germline a.a. 78 VK CDR3 a.a.11E6 105 VK L6germline a.a. 106 VK L15 germline a.a. 107 VK A27 germline a.a. 108 VKL18 germline a.a. 109 VK a.a. 11E6a

We claim:
 1. A monoclonal anti-PD-L1 antibody, or an antigen-bindingportion thereof, which cross-competes for binding to human PD-L1 with areference antibody, wherein the reference antibody comprises: (a) aheavy chain variable region comprising amino acids having the sequenceset forth in SEQ ID NO:1 and a light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO:11; (b) a heavychain variable region comprising amino acids having the sequence setforth in SEQ ID NO:2 and a light chain variable region comprising aminoacids having the sequence set forth in SEQ ID NO:12; (c) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:3 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:13; (d) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:4 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:14; (e) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:5 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:15; (f) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:6 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:16; (g) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:7 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:17; (h) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:8 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:18; (i) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:9 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:19; or (j) a heavy chainvariable region comprising amino acids having the sequence set forth inSEQ ID NO:10 and a light chain variable region comprising amino acidshaving the sequence set forth in SEQ ID NO:20; and wherein themonoclonal anti-PD-L1 antibody or antigen-binding portion thereofcomprises a heavy chain variable region (VH) and a light chain variableregion (VL), wherein the VH comprises a framework region exhibiting atleast 90% sequence identity to a framework region of the heavy chainvariable region of the reference antibody and/or the VL comprises aframework region exhibiting at least 90% sequence identity to aframework region of the light chain variable region of the referenceantibody.
 2. The monoclonal anti-PD-L1 antibody or antigen-bindingportion thereof of claim 1, wherein the framework region of the VHexhibits at least 95% sequence identity to the framework region of theheavy chain variable region of the reference antibody and/or theframework region of the VL exhibits at least 95% sequence identity tothe framework region of the light chain variable region of the referenceantibody.
 3. The monoclonal anti-PD-L1 antibody or antigen-bindingportion thereof of claim 1, wherein the VL exhibits at least 90%sequence identity to: (a) the light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO:11; (b) the lightchain variable region comprising amino acids having the sequence setforth in SEQ ID NO:12; (c) the light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO:13; (d) the lightchain variable region comprising amino acids having the sequence setforth in SEQ ID NO:14; (e) the light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO:15; (f) the lightchain variable region comprising amino acids having the sequence setforth in SEQ ID NO:16; (g) the light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO:17; (h) the lightchain variable region comprising amino acids having the sequence setforth in SEQ ID NO:18; (i) the light chain variable region comprisingamino acids having the sequence set forth in SEQ ID NO:19; or (j) thelight chain variable region comprising amino acids having the sequenceset forth in SEQ ID NO:20.
 4. The monoclonal anti-PD-L1 antibody orantigen-binding portion thereof of claim 1, wherein the VL comprises aFR1 selected from: (a) the FR1 of the light chain variable region havingthe amino acid sequence set forth in SEQ ID NO:11 or the FR1 of thelight chain variable region having the amino acid sequence set forth inSEQ ID NO:11 with one amino acid substitution; (b) the FR1 of the lightchain variable region having the amino acid sequence set forth in SEQ IDNO:12 or the FR1 of the light chain variable region having the aminoacid sequence set forth in SEQ ID NO:12 with one amino acidsubstitution; (c) the FR1 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:13 or the FR1 of the lightchain variable region having the amino acid sequence set forth in SEQ IDNO:13 with one amino acid substitution; (d) the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:14or the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:14 with one amino acid substitution; (e)the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:15 or the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:15with one amino acid substitution; (f) the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:16or the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:16 with one amino acid substitution; (g)the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:17 or the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:17with one amino acid substitution; (h) the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:18or the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:18 with one amino acid substitution; (i)the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:19 or the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:19with one amino acid substitution; and (j) the FR1 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:20or the FR1 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:20 with one amino acid substitution. 5.The monoclonal anti-PD-L1 antibody or antigen-binding portion thereof ofclaim 1, wherein the VL comprises a FR2 selected from: (a) the FR2 ofthe light chain variable region having the amino acid sequence set forthin SEQ ID NO:11 or the FR2 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:11 with one amino acidsubstitution; (b) the FR2 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:12 or the FR2 of the lightchain variable region having the amino acid sequence set forth in SEQ IDNO:12 with one amino acid substitution; (c) the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:13or the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:13 with one amino acid substitution; (d)the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:14 or the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:14with one amino acid substitution; (e) the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:15or the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:15 with one amino acid substitution; (f)the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:16 or the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:16with one amino acid substitution; (g) the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:17or the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:17 with one amino acid substitution; (h)the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:18 or the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:18with one amino acid substitution; (i) the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:19or the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:19 with one amino acid substitution; and(j) the FR2 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:20 or the FR2 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:20with one amino acid substitution.
 6. The monoclonal anti-PD-L1 antibodyor antigen-binding portion thereof of claim 1, wherein the VL comprisesa FR3 selected from: (a) the FR3 of the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:11 or the FR3 ofthe light chain variable region having the amino acid sequence set forthin SEQ ID NO:11 with one amino acid substitution; (b) the FR3 of thelight chain variable region having the amino acid sequence set forth inSEQ ID NO:12 or the FR3 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:12 with one amino acidsubstitution; (c) the FR3 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:13 or the FR3 of the lightchain variable region having the amino acid sequence set forth in SEQ IDNO:13 with one amino acid substitution; (d) the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:14or the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:14 with one amino acid substitution; (e)the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:15 or the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:15with one amino acid substitution; (f) the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:16or the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:16 with one amino acid substitution; (g)the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:17 or the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:17with one amino acid substitution; (h) the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:18or the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:18 with one amino acid substitution; (i)the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:19 or the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:19with one amino acid substitution; and (j) the FR3 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:20or the FR3 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:20 with one amino acid substitution. 7.The monoclonal anti-PD-L1 antibody or antigen-binding portion thereof ofclaim 1, wherein the VL comprises a FR4 selected from: (a) the FR4 ofthe light chain variable region having the amino acid sequence set forthin SEQ ID NO:11 or the FR4 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:11 with one amino acidsubstitution; (b) the FR4 of the light chain variable region having theamino acid sequence set forth in SEQ ID NO:12 or the FR4 of the lightchain variable region having the amino acid sequence set forth in SEQ IDNO:12 with one amino acid substitution; (c) the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:13or the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:13 with one amino acid substitution; (d)the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:14 or the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO 14with one amino acid substitution; (e) the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:15or the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:15 with one amino acid substitution; (f)the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:16 or the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:16with one amino acid substitution; (g) the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:17or the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:17 with one amino acid substitution; (h)the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:18 or the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:18with one amino acid substitution; (i) the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:19or the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:19 with one amino acid substitution; and(j) the FR4 of the light chain variable region having the amino acidsequence set forth in SEQ ID NO:20 or the FR4 of the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:20with one amino acid substitution.
 8. The monoclonal anti-PD-L1 antibodyor antigen-binding portion thereof of claim 1, wherein the VH comprisesa FR1 selected from: (a) the FR1 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:1 or the FR1 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:1 with one amino acid substitution; (b) the FR1 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:2 or the FR1 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:2 with one amino acidsubstitution; (c) the FR1 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:3 or the FR1 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:3 with one amino acid substitution; (d) the FR1 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:4or the FR1 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:4 with one amino acid substitution; (e)the FR1 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:5 or the FR1 of the heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO:5 with oneamino acid substitution; (f) the FR1 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:6 or the FR1 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:6 with one amino acid substitution; (g) the FR1 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:7 or the FR1 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:7 with one amino acidsubstitution; (h) the FR1 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:8 or the FR1 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:8 with one amino acid substitution; (i) the FR1 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:9or the FR1 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:9 with one amino acid substitution; and(j) the FR1 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:10 or the FR1 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:10with one amino acid substitution.
 9. The monoclonal anti-PD-L1 antibodyor antigen-binding portion thereof of claim 1, wherein the VH comprisesa FR2 selected from: (a) the FR2 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:1 or the FR2 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:1 with one amino acid substitution; (b) the FR2 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:2 or the FR2 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:2 with one amino acidsubstitution; (c) the FR2 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:3 or the FR2 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:3 with one amino acid substitution; (d) the FR2 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:4or the FR2 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:4 with one amino acid substitution; (e)the FR2 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:5 or the FR2 of the heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO:5 with oneamino acid substitution; (f) the FR2 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:6 or the FR2 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:6 with one amino acid substitution; (g) the FR2 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:7 or the FR2 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:7 with one amino acidsubstitution; (h) the FR2 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:8 or the FR2 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:8 with one amino acid substitution; (i) the FR2 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:9or the FR2 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:9 with one amino acid substitution; and(j) the FR2 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:10 or the FR2 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:10with one amino acid substitution.
 10. The monoclonal anti-PD-L1 antibodyor antigen-binding portion thereof of claim 1, wherein the VH comprisesa FR3 selected from: (a) the FR3 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:1 or the FR3 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:1 with one amino acid substitution; (b) the FR3 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:2 or the FR3 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:2 with one amino acidsubstitution; (c) the FR3 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:3 or the FR3 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:3 with one amino acid substitution; (d) the FR3 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:4or the FR3 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:4 with one amino acid substitution; (e)the FR3 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:5 or the FR3 of the heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO:5 with oneamino acid substitution; (f) the FR3 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:6 or the FR3 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:6 with one amino acid substitution; (g) the FR3 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:7 or the FR3 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:7 with one amino acidsubstitution; (h) the FR3 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:8 or the FR3 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:8 with one amino acid substitution; (i) the FR3 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:9or the FR3 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:9 with one amino acid substitution; and(j) the FR3 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:10 or the FR3 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:10with one amino acid substitution.
 11. The monoclonal anti-PD-L1 antibodyor antigen-binding portion thereof of claim 1, wherein the VH comprisesa FR4 selected from: (a) the FR4 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:1 or the FR4 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:1 with one amino acid substitution; (b) the FR4 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:2 or the FR4 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:2 with one amino acidsubstitution; (c) the FR4 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:3 or the FR4 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:3 with one amino acid substitution; (d) the FR4 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:4or the FR4 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:4 with one amino acid substitution; (e)the FR4 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:5 or the FR4 of the heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO:5 with oneamino acid substitution; (f) the FR4 of the heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:6 or the FR4 ofthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO:6 with one amino acid substitution; (g) the FR4 of theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:7 or the FR4 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:7 with one amino acidsubstitution; (h) the FR4 of the heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:8 or the FR4 of the heavychain variable region having the amino acid sequence set forth in SEQ IDNO:8 with one amino acid substitution; (i) the FR4 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:9or the FR4 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:9 with one amino acid substitution; and(j) the FR4 of the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO:10 or the FR4 of the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:10with one amino acid substitution.
 12. The monoclonal anti-PD-L1 antibodyor antigen-binding portion thereof of claim 1, wherein the VH is of ahuman IgG1, a human IgG2, a human IgG3, or a human IgG4 isotype.
 13. Themonoclonal anti-PD-L1 antibody or antigen-binding portion thereof ofclaim 12, wherein the VH is of a human IgG4 isotype.
 14. The monoclonalanti-PD-L1 antibody or antigen-binding portion thereof of claim 12,wherein the VH is of a human IgG1 isotype and is modified by replacingat least one amino acid residue with a different amino acid residue toalter one or more effector functions of the monoclonal anti-PD-L1antibody or antigen-binding portion thereof.
 15. The monoclonalanti-PD-L1 antibody or antigen-binding portion thereof of claim 1, whichbinds to human PD-L1 with a KD of 5×10⁻⁹ M or less.
 16. The monoclonalanti-PD-L1 antibody or antigen-binding portion thereof of claim 1, whichbinds to human PD-L1 with a KD of 2×10⁻⁹ M or less.
 17. The monoclonalanti-PD-L1 antibody or antigen-binding portion thereof of claim 1, whichbinds to human PD-L1 with a KD of 1×10⁻⁹ M or less.
 18. The monoclonalanti-PD-L1 antibody or antigen-binding portion thereof of claim 1,wherein the reference antibody binds to human PD-L1 with a KD of about5×10⁻⁹ M.
 19. The monoclonal anti-PD-L1 antibody or antigen-bindingportion thereof of claim 1, wherein the reference antibody binds tohuman PD-L1 with a KD of 2×10⁻⁹ M or less.
 20. The monoclonal anti-PD-L1antibody or antigen-binding portion thereof of claim 1, wherein thereference antibody binds to human PD-L1 with a KD of 1×10⁻⁹ M or less.21. The monoclonal anti-PD-L1 antibody or antigen-binding portionthereof of claim 1, which exhibits the following properties: (a) bindswith high affinity to human PD-L1 with a KD of about 2×10⁻⁹ M or less asdetermined by surface plasmon resonance analysis using a Biacore system;(b) inhibits binding of PD-L1 to a PD-1 receptor; (c) augments T cellproliferation, IFN-γ and IL-2 secretion in mixed lymphocyte reactionassays; (d) stimulates antibody responses; and (e) reverses thesuppressive effect of T regulatory cells on T cell effector cells,dendritic cells, or T cell effector cells and dendritic cells.
 22. Themonoclonal anti-PD-L1 antibody or antigen-binding portion thereof ofclaim 1, which is a human or a humanized antibody or a portion thereof.23. The monoclonal anti-PD-L1 antibody or antigen-binding portionthereof of claim 1, comprising: (a) the VH which comprises amino acidshaving the sequence set forth in SEQ ID NO:1 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:11 or a conservative sequencemodification thereof; (b) the VH which comprises amino acids having thesequence set forth in SEQ ID NO:2 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:12 or a conservative sequencemodification thereof; (c) the VH which comprises amino acids having thesequence set forth in SEQ ID NO:6 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:16 or a conservative sequencemodification thereof; (d) the VH which comprises amino acids having thesequence set forth in SEQ ID NO:7 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:17 or a conservative sequencemodification thereof; (e) the VH which comprises amino acids having thesequence set forth in SEQ ID NO:8 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:18 or a conservative sequencemodification thereof; (f) the VH which comprises amino acids having thesequence set forth in SEQ ID NO:9 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:19 or a conservative sequencemodification thereof; or (g) the VH which comprises amino acids havingthe sequence set forth in SEQ ID NO:10 or a conservative sequencemodification thereof and the VL which comprises amino acids having thesequence set forth in SEQ ID NO:20 or a conservative sequencemodification thereof, wherein the conservative sequence modificationcomprises one or more additions, deletions, or conservative amino acidsubstitutions, and wherein the antibodies comprising a conservativesequence modification retain the functional characteristics ofunmodified antibodies.
 24. The monoclonal anti-PD-L1 antibody orantigen-binding portion thereof of claim 1, comprising the VH whichcomprises amino acids having the sequence set forth in SEQ ID NO:2 or aconservative sequence modification thereof and the VL which comprisesamino acids having the sequence set forth in SEQ ID NO:12 or aconservative sequence modification thereof, wherein the conservativesequence modification comprises one or more additions, deletions, orconservative amino acid substitutions.
 25. The monoclonal anti-PD-L1antibody or antigen-binding portion thereof of claim 23, wherein theconservative sequence modification comprises a modification of one ormore residues within one or more CDR regions, within one or moreframework regions, or within one or more CDR regions and one or moreframework regions.
 26. The monoclonal anti-PD-L1 antibody orantigen-binding portion thereof of claim 23, wherein no more than threeamino acid residues within a CDR region are modified.
 27. A compositioncomprising the monoclonal anti-PD-L1 antibody or antigen-binding portionthereof of claim 1, formulated together with a pharmaceuticallyacceptable carrier.
 28. The composition of claim 27, wherein thepharmaceutically acceptable carrier is suitable for intravenousadministration.
 29. A kit comprising the composition of claim 27, alabel indicating the intended use of the contents of the kit andinstructions for use.
 30. The kit of claim A, further comprising atleast one additional anti-neoplastic agent.